Methods and compositions for targeted cleavage and recombination

ABSTRACT

Disclosed herein are methods and compositions for targeted cleavage of a genomic sequence, targeted alteration of a genomic sequence, and targeted recombination between a genomic region and an exogenous polynucleotide homologous to the genomic region. The compositions include fusion proteins comprising a cleavage domain (or cleavage half-domain) and an engineered zinc finger domain and polynucleotides encoding same. Methods for targeted cleavage include introduction of such fusion proteins, or polynucleotides encoding same, into a cell. Methods for targeted recombination additionally include introduction of an exogenous polynucleotide homologous to a genomic region into cells comprising the disclosed fusion proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/912,932 filed Aug. 6, 2004, which claims the benefit of the followingU.S. provisional patent applications: 60/493,931 filed Aug. 8, 2003;60/518,253 filed Nov. 7, 2003; 60/530,541 filed Dec. 18, 2003;60/542,780 filed Feb. 5, 2004; 60/556,831 filed Mar. 26, 2004 and60/575,919 filed Jun. 1, 2004; the disclosures of which are incorporatedby reference in their entireties for all purposes.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

TECHNICAL FIELD

The present disclosure is in the field of genome engineering andhomologous recombination.

BACKGROUND

A major area of interest in genome biology, especially in light of thedetermination of the complete nucleotide sequences of a number ofgenomes, is the targeted alteration of genome sequences. To provide butone example, sickle cell anemia is caused by mutation of a singlenucleotide pair in the human β-globin gene. Thus, the ability to convertthe endogenous genomic copy of this mutant nucleotide pair to thewild-type sequence in a stable fashion and produce normal β-globin wouldprovide a cure for sickle cell anemia.

Attempts have been made to alter genomic sequences in cultured cells bytaking advantage of the natural phenomenon of homologous recombination.See, for example, Capecchi (1989) Science 244:1288-1292; U.S. Pat. Nos.6,528,313 and 6,528,314. If a polynucleotide has sufficient homology tothe genomic region containing the sequence to be altered, it is possiblefor part or all of the sequence of the polynucleotide to replace thegenomic sequence by homologous recombination. However, the frequency ofhomologous recombination under these circumstances is extremely low.Moreover, the frequency of insertion of the exogenous polynucleotide atgenomic locations that lack sequence homology exceeds the frequency ofhomologous recombination by several orders of magnitude.

The introduction of a double-stranded break into genomic DNA, in theregion of the genome bearing homology to an exogenous polynucleotide,has been shown to stimulate homologous recombination at this site byseveral thousand-fold in cultured cells. Rouet et al. (1994) Mol. Cell.Biol. 14:8096-8106; Choulika et al. (1995) Mol. Cell. Biol.15:1968-1973; Donoho et al. (1998) Mol. Cell. Biol. 18:4070-4078. Seealso Johnson et al. (2001) Biochem. Soc. Trans. 29:196-201; and Yanez etal. (1998) Gene Therapy 5:149-159. In these methods, DNA cleavage in thedesired genomic region was accomplished by inserting a recognition sitefor a meganuclease (i.e., an endonuclease whose recognition sequence isso large that it does not occur, Or occurs only rarely, in the genome ofinterest) into the desired genomic region.

However, meganuclease cleavage-stimulated homologous recombinationrelies on either the fortuitous presence of, or the directed insertionof, a suitable meganuclease recognition site in the vicinity of thegenomic region to be altered. Since meganuclease recognition sites arerare (or nonexistent) in a typical mammalian genome, and insertion of asuitable meganuclease recognition site is plagued with the samedifficulties as associated with other genomic alterations, these methodsare not broadly applicable.

Thus, there remains a need for compositions and methods for targetedalteration of sequences in any genome.

SUMMARY

The present disclosure provides compositions and methods for targetedcleavage of cellular chromatin in a region of interest and/or homologousrecombination at a predetermined region of interest in cells. Cellsinclude cultured cells, cells in an organism and cells that have beenremoved from an organism for treatment in cases where the cells and/ortheir descendants will be returned to the organism after treatment. Aregion of interest in cellular chromatin can be, for example, a genomicsequence or portion thereof. Compositions include fusion polypeptidescomprising an engineered zinc finger binding domain (e.g., a zinc fingerbinding domain having a novel specificity) and a cleavage domain, andfusion polypeptides comprising an engineered zinc finger binding domainand a cleavage half-domain. Cleavage domains and cleavage half domainscan be obtained, for example, from various restriction endonucleasesand/or homing endonucleases.

Cellular chromatin can be present in any type of cell including, but notlimited to, prokaryotic and eukaryotic cells, fungal cells, plant cells,animal cells, mammalian cells, primate cells and human cells.

In one aspect, a method for cleavage of cellular chromatin in a regionof interest (e.g., a method for targeted cleavage of genomic sequences)is provided, the method comprising: (a) selecting a first sequence inthe region of interest; (b) engineering a first zinc finger bindingdomain to bind to the first sequence; and (c) expressing a first fusionprotein in the cell, the first fusion protein comprising the firstengineered zinc finger binding domain and a cleavage domain; wherein thefirst fusion protein binds to the first sequence and the cellularchromatin is cleaved in the region of interest. The site of cleavage canbe coincident with the sequence to which the fusion protein binds, or itcan be adjacent (e.g., separated from the near edge of the binding siteby 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or morenucleotides). A fusion protein can be expressed in a cell, e.g., bydelivering the fusion protein to the cell or by delivering apolynucleotide encoding the fusion protein to a cell, wherein thepolynucleotide, if DNA, is transcribed, and an RNA molecule delivered tothe cell or a transcript of a DNA molecule delivered to the cell istranslated, to generate the fusion protein. Methods for polynucleotideand polypeptide delivery to cells are presented elsewhere in thisdisclosure.

In certain embodiments, the cleavage domain may comprise two cleavagehalf-domains that are covalently linked in the same polypeptide. The twocleavage half-domains can be derived from the same endonuclease or fromdifferent endonucleases.

In additional embodiments, targeted cleavage of cellular chromatin in aregion of interest is achieved by expressing two fusion proteins in acell, each fusion protein comprising a zinc finger binding domain and acleavage half-domain. One or both of the zinc finger binding domains ofthe fusion proteins can be engineered to bind to a target sequence inthe vicinity of the intended cleavage site. If expression of the fusionproteins is by polynucleotide delivery, each of the two fusion proteinscan be encoded by a separate polynucleotide, or a single polynucleotidecan encode both fusion proteins.

Accordingly, a method for cleaving cellular chromatin in a region ofinterest can comprise (a) selecting a first sequence in the region ofinterest; (b) engineering a first zinc finger binding domain to bind tothe first sequence; (c) expressing a first fusion protein in the cell,the first fusion protein comprising the first zinc finger binding domainand a first cleavage half-domain; and (d) expressing a second fusionprotein in the cell, the second fusion protein comprising a second zincfinger binding domain and a second cleavage half-domain, wherein thefirst fusion protein binds to the first sequence, and the second fusionprotein binds to a second sequence located between 2 and 50 nucleotidesfrom the first sequence, thereby positioning the cleavage half-domainssuch that the cellular chromatin is cleaved in the region of interest.

In certain embodiments, binding of the first and second fusion proteinspositions the cleavage half-domains such that a functional cleavagedomain is reconstituted.

In certain embodiments, the second zinc finger binding domain isengineered to bind to the second sequence. In further embodiments, thefirst and second cleavage half-domains are derived from the sameendonuclease, which can be, for example, a restriction endonuclease(e.g., a Type IIS restriction endonuclease such as Fok I) or a homingendonuclease.

In other embodiments, any of the methods described herein may comprise(a) selecting first and second sequences in a region of interest,wherein the first and second sequences are between 2 and 50 nucleotidesapart; (b) engineering a first zinc finger binding domain to bind to thefirst sequence; (c) engineering a second zinc finger binding domain tobind to the second sequence; (d) expressing a first fusion protein inthe cell, the first fusion protein comprising the first engineered zincfinger binding domain and a first cleavage half-domain; (e) expressing asecond fusion protein in the cell, the second fusion protein comprisingthe second engineered zinc finger binding domain and a second cleavagehalf-domain; wherein the first fusion protein binds to the firstsequence and the second fusion protein binds to the second sequence,thereby positioning the first and second cleavage half-domains such thatthe cellular chromatin is cleaved in the region of interest.

In certain embodiments, the first and second cleavage half-domains arederived from the same endonuclease, for example, a Type IIS restrictionendonuclease, for example, Fok I. In additional embodiments, cellularchromatin is cleaved at one or more sites between the first and secondsequences to which the fusion proteins bind.

In further embodiments, a method for cleavage of cellular chromatin in aregion of interest comprises (a) selecting the region of interest; (b)engineering a first zinc finger binding domain to bind to a firstsequence in the region of interest; (c) providing a second zinc fingerbinding domain which binds to a second sequence in the region ofinterest, wherein the second sequence is located between 2 and 50nucleotides from the first sequence; (d) expressing a first fusionprotein in the cell, the first fusion protein comprising the first zincfinger binding domain and a first cleavage half-domain; and (e)expressing a second fusion protein in the cell, the second fusionprotein comprising the second zinc finger binding domain and a secondcleavage half domain; wherein the first fusion protein binds to thefirst sequence, and the second fusion protein binds to the secondsequence, thereby positioning the cleavage half-domains such that thecellular chromatin is cleaved in the region of interest.

In any of the methods described herein, the first and second cleavagehalf-domains may be derived from the same endonuclease or from differentendonucleases. In additional embodiments, the second zinc finger bindingdomain is engineered to bind to the second sequence.

If one or more polynucleotides encoding the fusion proteins areintroduced into the cell, an exemplary method for targeted cleavage ofcellular chromatin in a region of interest comprises (a) selecting theregion of interest; (b) engineering a first zinc finger binding domainto bind to a first sequence in the region of interest; (c) providing asecond zinc finger binding domain which binds to a second sequence inthe region of interest, wherein the second sequence is located between 2and 50 nucleotides from the first sequence; and (d) contacting a cellwith (i) a first polynucleotide encoding a first fusion protein, thefusion protein comprising the first zinc finger binding domain and afirst cleavage half-domain, and (ii) a second polynucleotide encoding asecond fusion protein, the fusion protein comprising the second zincfinger binding domain and a second cleavage half domain; wherein thefirst and second fusion proteins are expressed, the first fusion proteinbinds to the first sequence and the second fusion protein binds to thesecond sequence, thereby positioning the cleavage half-domains such thatthe cellular chromatin is cleaved in the region of interest. In avariation of this method, a cell is contacted with a singlepolynucleotide which encodes both fusion proteins.

For any of the aforementioned methods, the cellular chromatin can be ina chromosome, episome or organellar genome. In addition, in any of themethods described herein, at least one zinc finger binding domain isengineered, for example by design or selection methods.

Similarly, for any of the aforementioned methods, the cleavage halfdomain can be derived from, for example, a homing endonuclease or arestriction endonuclease, for example, a Type IIS restrictionendonuclease. An exemplary Type IIS restriction endonuclease is Fok I.

For any of the methods of targeted cleavage, targeted mutagenesis and/ortargeted recombination disclosed herein utilizing fusion proteinscomprising a cleavage half-domain, the near edges of the binding sitesof the fusion proteins can be separated by 5 or 6 base pairs. In theseembodiments, the binding domain and the cleavage domain of the fusionproteins can be separated by a linker of 4 amino acid residues.

In certain embodiments, it is possible to obtain increased cleavagespecificity by utilizing fusion proteins in which one or both cleavagehalf-domains contains an alteration in the amino acid sequence of thedimerization interface.

Targeted mutagenesis of a region of interest in cellular chromatin canoccur when a targeted cleavage event, as describe above, is followed bynon-homologous end joining (NHEJ). Accordingly, methods for alterationof a first nucleotide sequence in a region of interest in cellularchromatin are provided, wherein the methods comprise the steps of (a)engineering a first zinc finger binding domain to bind to a secondnucleotide sequence in the region of interest, wherein the secondsequence comprises at least 9 nucleotides; (b) providing a second zincfinger binding domain to bind to a third nucleotide sequence, whereinthe third sequence comprises at least 9 nucleotides and is locatedbetween 2 and 50 nucleotides from the second sequence; (c) expressing afirst fusion protein in the cell, the first fusion protein comprisingthe first zinc finger binding domain and a first cleavage half-domain;and (d) expressing a second fusion protein in the cell, the secondfusion protein comprising the second zinc finger binding domain and asecond cleavage half domain; wherein the first fusion protein binds tothe second sequence, and the second fusion protein binds to the thirdsequence, thereby positioning the cleavage half-domains such that thecellular chromatin is cleaved in the region of interest and the cleavagesite is subjected to non-homologous end joining.

Targeted mutations resulting from the aforementioned method include, butare not limited to, point mutations (i.e., conversion of a single basepair to a different base pair), substitutions (i.e., conversion of aplurality of base pairs to a different sequence of identical length),insertions or one or more base pairs, deletions of one or more basepairs and any combination of the aforementioned sequence alterations.

Methods for targeted recombination (for, e.g., alteration or replacementof a sequence in a chromosome or a region of interest in cellularchromatin) are also provided. For example, a mutant genomic sequence canbe replaced by a wild-type sequence, e.g., for treatment of geneticdisease or inherited disorders. In addition, a wild-type genomicsequence can be replaced by a mutant sequence, e.g., to prevent functionof an oncogene product or a product of a gene involved in aninappropriate inflammatory response. Furthermore, one allele of a genecan be replaced by a different allele.

In such methods, one or more targeted nucleases create a double-strandedbreak in cellular chromatin at a predetermined site, and a donorpolynucleotide, having homology to the nucleotide sequence of thecellular chromatin in the region of the break, is introduced into thecell. Cellular DNA repair processes are activated by the presence of thedouble-stranded break and the donor polynucleotide is used as a templatefor repair of the break, resulting in the introduction of all or part ofthe nucleotide sequence of the donor into the cellular chromatin. Thus afirst sequence in cellular chromatin can be altered and, in certainembodiments, can be converted into a sequence present in a donorpolynucleotide.

In this context, the use of the terms “replace” or “replacement” can beunderstood to represent replacement of one nucleotide sequence byanother, (i.e., replacement of a sequence in the informational sense),and does not necessarily require physical or chemical replacement of onepolynucleotide by another.

Accordingly, in one aspect, a method for replacement of a region ofinterest in cellular chromatin (e.g., a genomic sequence) with a firstnucleotide sequence is provided, the method comprising: (a) engineeringa zinc finger binding domain to bind to a second sequence in the regionof interest; (b) expressing a fusion protein in a cell, the fusionprotein comprising the zinc finger binding domain and a cleavage domain;and (c) contacting the cell with a polynucleotide comprising the firstnucleotide sequence; wherein the fusion protein binds to the secondsequence such that the cellular chromatin is cleaved in the region ofinterest and a nucleotide sequence in the region of interest is replacedwith the first nucleotide sequence. Generally, cellular chromatin iscleaved in the region of interest at or adjacent to the second sequence.In further embodiments, the cleavage domain comprises two cleavagehalf-domains, which can be derived from the same or from differentnucleases.

In addition, a method for replacement of a region of interest incellular chromatin (e.g., a genomic sequence) with a first nucleotidesequence is provided, the method comprising: (a) engineering a firstzinc finger binding domain to bind to a second sequence in the region ofinterest; (b) providing a second zinc finger binding domain to bind to athird sequence in the region of interest; (c) expressing a first fusionprotein in a cell, the first fusion protein comprising the first zincfinger binding domain and a first cleavage half-domain; (d) expressing asecond fusion protein in the cell, the second fusion protein comprisingthe second zinc finger binding domain and a second cleavage half-domain;and (e) contacting the cell with a polynucleotide comprising the firstnucleotide sequence; wherein the first fusion protein binds to thesecond sequence and the second fusion protein binds to the thirdsequence, thereby positioning the cleavage half-domains such that thecellular chromatin is cleaved in the region of interest and a nucleotidesequence in the region of interest is replaced with the first nucleotidesequence. Generally, cellular chromatin is cleaved in the region ofinterest at a site between the second and third sequences.

Additional methods for replacement of a region of interest in cellularchromatin (e.g., a genomic sequence) with a first nucleotide sequencecomprise: (a) selecting a second sequence, wherein the second sequenceis in the region of interest and has a length of at least 9 nucleotides;(b) engineering a first zinc finger binding domain to bind to the secondsequence; (c) selecting a third sequence, wherein the third sequence hasa length of at least 9 nucleotides and is located between 2 and 50nucleotides from the second sequence; (d) providing a second zinc fingerbinding domain to bind to the third sequence; (e) expressing a firstfusion protein in a cell, the first fusion protein comprising the firstzinc finger binding domain and a first cleavage half-domain; (f)expressing a second fusion protein in the cell, the second fusionprotein comprising the second zinc finger binding domain and a secondcleavage half-domain; and (g) contacting the cell with a polynucleotidecomprising the first nucleotide sequence; wherein the first fusionprotein binds to the second sequence and the second fusion protein bindsto the third sequence, thereby positioning the cleavage half-domainssuch that the cellular chromatin is cleaved in the region of interestand a nucleotide sequence in the region of interest is replaced with thefirst nucleotide sequence. Generally, cellular chromatin is cleaved inthe region of interest at a site between the second and third sequences.

In another aspect, methods for targeted recombination are provided inwhich, a first nucleotide sequence, located in a region of interest incellular chromatin, is replaced with a second nucleotide sequence. Themethods comprise (a) engineering a first zinc finger binding domain tobind to a third sequence in the region of interest; (b) providing asecond zinc finger binding domain to bind to a fourth sequence; (c)expressing a first fusion protein in a cell, the fusion proteincomprising the first zinc finger binding domain, and a first cleavagehalf-domain; (d) expressing a second fusion protein in the cell, thesecond fusion protein comprising the second zinc finger binding domainand a second cleavage half-domain; and (e) contacting a cell with apolynucleotide comprising the second nucleotide sequence; wherein thefirst fusion protein binds to the third sequence and the second fusionprotein binds to the fourth sequence, thereby positioning the cleavagehalf-domains such that the cellular chromatin is cleaved in the regionof interest and the first nucleotide sequence is replaced with thesecond nucleotide sequence.

In additional embodiments, a method for alteration of a first nucleotidesequence in a region of interest in cellular chromatin is provided, themethod comprising the steps of (a) engineering a first zinc fingerbinding domain to bind to a second nucleotide sequence in the region ofinterest, wherein the second sequence comprises at least 9 nucleotides;(b) providing a second zinc finger binding domain to bind to a thirdnucleotide sequence, wherein the third sequence comprises at least 9nucleotides and is located between 2 and 50 nucleotides from the secondsequence; (c) expressing a first fusion protein in the cell, the firstfusion protein comprising the first zinc finger binding domain and afirst cleavage half-domain; (d) expressing a second fusion protein inthe cell, the second fusion protein comprising the second zinc fingerbinding domain and a second cleavage half domain; and (e) contacting thecell with a polynucleotide comprising a fourth nucleotide sequence,wherein the fourth nucleotide sequence is homologous but non-identicalwith the first nucleotide sequence; wherein the first fusion proteinbinds to the second sequence, and the second fusion protein binds to thethird sequence, thereby positioning the cleavage half-domains such thatthe cellular chromatin is cleaved in the region of interest and thefirst nucleotide sequence is altered. In certain embodiments, the firstnucleotide sequence is converted to the fourth nucleotide sequence. Inadditional embodiments, the second and third nucleotide sequences (i.e.,the binding sites for the fusion proteins) are present in thepolynucleotide comprising the fourth nucleotide sequence (i.e., thedonor polynucleotide) and the polynucleotide comprising the fourthnucleotide sequence is cleaved.

In the aforementioned methods for targeted recombination, the bindingsites for the fusion proteins (i.e., the third and fourth sequences) cancomprise any number of nucleotides. Preferably, they are at least ninenucleotides in length, but they can also be larger (e.g., 10, 11, 12,13, 14, 15, 16, 17, 18 and up to 100 nucleotides, including any integralvalue between 9 and 100 nucleotides); moreover the third and fourthsequences need not be the same length. The distance between the bindingsites (i.e., the length of nucleotide sequence between the third andfourth sequences) can be any integral number of nucleotide pairs between2 and 50, (e.g., 5 or 6 base pairs) as measured from the near end of onebinding site to the near end of the other binding site.

In the aforementioned methods for targeted recombination, cellularchromatin can be cleaved at a site located between the binding sites ofthe two fusion proteins. In certain embodiments, the binding sites areon opposite DNA strands. Moreover, expression of the fusion proteins inthe cell can be accomplished either by introduction of the proteins intothe cell or by introduction of one or more polynucleotides into thecell, which are optionally transcribed (if the polynucleotide is DNA),and the transcript(s) translated, to produce the fusion proteins. Forexample, two polynucleotides, each comprising sequences encoding one ofthe two fusion proteins, can be introduced into a cell. Alternatively, asingle polynucleotide comprising sequences encoding both fusion proteinscan be introduced into the cell.

Thus, in one embodiment, a method for replacement of a region ofinterest in cellular chromatin (e.g., a genomic sequence) with a firstnucleotide sequence comprises: (a) engineering a first zinc fingerbinding domain to bind to a second sequence in the region of interest;(b) providing a second zinc finger binding domain to bind to a thirdsequence; and (c) contacting a cell with:

(i) a first polynucleotide comprising the first nucleotide sequence;

(ii) a second polynucleotide encoding a first fusion protein, the firstfusion protein comprising the first zinc finger binding domain and afirst cleavage half-domain; and

(iii) a third polynucleotide encoding a second fusion protein, thesecond fusion protein comprising the second zinc finger binding domainand a second cleavage half-domain;

wherein the first and second fusion proteins are expressed, the firstfusion protein binds to the second sequence and the second fusionprotein binds to the third sequence, thereby positioning the cleavagehalf-domains such that the cellular chromatin is cleaved in the regionof interest; and the region of interest is replaced with the firstnucleotide sequence.

In the preferred embodiments of methods for targeted recombinationand/or replacement and/or alteration of a sequence in a region ofinterest in cellular chromatin, a chromosomal sequence is altered byhomologous recombination with an exogenous “donor” nucleotide sequence.Such homologous recombination is stimulated by the presence of adouble-stranded break in cellular chromatin, if sequences homologous tothe region of the break are present. Double-strand breaks in cellularchromatin can also stimulate cellular mechanisms of non-homologous endjoining.

In any of the methods described herein, the first nucleotide sequence(the “donor sequence”) can contain sequences that are homologous, butnot identical, to genomic sequences in the region of interest, therebystimulating homologous recombination to insert a non-identical sequencein the region of interest. Thus, in certain embodiments, portions of thedonor sequence that are homologous to sequences in the region ofinterest exhibit between about 80 to 99% (or any integer therebetween)sequence identity to the genomic sequence that is replaced. In otherembodiments, the homology between the donor and genomic sequence ishigher than 99%, for example if only 1 nucleotide differs as betweendonor and genomic sequences of over 100 contiguous base pairs. Incertain cases, a non-homologous portion of the donor sequence cancontain sequences not present in the region of interest, such that newsequences are introduced into the region of interest. In theseinstances, the non-homologous sequence is generally flanked by sequencesof 50-1,000 base pairs (or any integral value therebetween) or anynumber of base pairs greater than 1,000, that are homologous oridentical to sequences in the region of interest. In other embodiments,the donor sequence is non-homologous to the first sequence, and isinserted into the genome by non-homologous recombination mechanisms.

In methods for targeted recombination and/or replacement and/oralteration of a sequence of interest in cellular chromatin, the firstand second cleavage half-domains can be derived from the sameendonuclease or from different endonucleases. Endonucleases include, butare not limited to, homing endonucleases and restriction endonucleases.Exemplary restriction endonucleases are Type IIS restrictionendonucleases; an exemplary Type IIS restriction endonuclease is Fok I.

The region of interest can be in a chromosome, episome or organellargenome. The region of interest can comprise a mutation, which canreplaced by a wild type sequence (or by a different mutant sequence), orthe region of interest can contain a wild-type sequence that is replacedby a mutant sequence or a different allele. Mutations include, but arenot limited to, point mutations (transitions, transversions), insertionsof one or more nucleotide pairs, deletions of one or more nucleotidepairs, rearrangements, inversions and translocations. Mutations canchange the coding sequence, introduce premature stop codon(s) and/ormodify the frequency of a repetitive sequence motif (e.g., trinucleotiderepeat) in a gene. For applications in which targeted recombination isused to replace a mutant sequence, cellular chromatin is generallycleaved at a site located within 100 nucleotides on either side of themutation, although cleavage sites located up to 6-10 kb from the site ofa mutation can also be used.

In any of the methods described herein, the second zinc finger bindingdomain can be engineered, for example designed and/or selected.

Further, the donor polynucleotide can be DNA or RNA, can be linear orcircular, and can be single-stranded or double-stranded. It can bedelivered to the cell as naked nucleic acid, as a complex with one ormore delivery agents (e.g., liposomes, poloxamers) or contained in aviral delivery vehicle, such as, for example, an adenovirus or anadeno-associated Virus (AAV). Donor sequences can range in length from10 to 1,000 nucleotides (or any integral value of nucleotidestherebetween) or longer.

Similarly, polynucleotides encoding fusions between a zinc fingerbinding domain and a cleavage domain or half-domain can be DNA or RNA,can be linear or circular, and can be single-stranded ordouble-stranded. They can be delivered to the cell as naked nucleicacid, as a complex with one or more delivery agents (e.g., liposomes,poloxamers) or contained in a viral delivery vehicle, such as, forexample, an adenovirus or an adeno-associated virus (AAV). Apolynucleotide can encode one or more fusion proteins.

In the methods for targeted recombination, as with the methods fortargeted cleavage, a cleavage domain or half-domain can derived from anynuclease, e.g., a homing endonuclease or a restriction endonuclease, inparticular, a Type IIS restriction endonuclease. Cleavage half-domainscan derived from the same or from different endonucleases. An exemplarysource, from which a cleavage half-domain can be derived, is the TypeIIS restriction endonuclease Fok I.

In certain embodiments, the frequency of homologous recombination can beenhanced by arresting the cells in the G2 phase of the cell cycle and/orby activating the expression of one or more molecules (protein, RNA)involved in homologous recombination and/or by inhibiting the expressionor activity of proteins involved in non-homologous end-joining.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence, in double-stranded form, of aportion of the human hSMC1L1 gene encoding the amino-terminal portion ofthe protein (SEQ ID NO:1) and the encoded amino acid sequence (SEQ IDNO:2). Target sequences for the hSMC1-specific ZFPs are underlined (oneon each DNA strand).

FIG. 2 shows a schematic diagram of a plasmid encoding a ZFP-FokI fusionfor targeted cleavage of the hSMC1 gene.

FIG. 3 A-D show a schematic diagram of the hSMC1 gene. FIG. 3A shows aschematic of a portion of the human X chromosome which includes thehSMC1 gene. FIG. 3B shows a schematic of a portion of the hSMC1 geneincluding the upstream region (left of +1), the first exon (between +1and the right end of the arrow labeled “SMC1 coding sequence”) and aportion of the first intron. Locations of sequences homologous to theinitial amplification primers and to the chromosome-specific primer (seeTable 3) are also provided. FIG. 3C shows the nucleotide sequence of thehuman X chromosome in the region of the SMC1 initiation codon (SEQ IDNO: 3), the encoded amino acid sequence (SEQ ID NO: 4), and the targetsites for the SMC1-specific zinc finger proteins. FIG. 3D shows thesequence of the corresponding region of the donor molecule (SEQ ID NO:5), with differences between donor and chromosomal sequences underlined.Sequences contained in the donor-specific amplification primer (Table 3)are indicated by double underlining.

FIG. 4 shows a schematic diagram of the hSMC1 donor construct.

FIG. 5 shows PCR analysis of DNA from transfected HEK293 cells. Fromleft, the lanes show results from cells transfected with a plasmidencoding GFP (control plasmid), cells transfected with two plasmids,each of which encodes one of the two hSMC1-specific ZFP-FokI fusionproteins (ZFPs only), cells transfected with two concentrations of thehSMC1 donor plasmid (donor only), and cells transfected with the twoZFP-encoding plasmids and the donor plasmid (ZFPs+donor). See Example 1for details.

FIG. 6 shows the nucleotide sequence of an amplification product derivedfrom a mutated hSMC1 gene (SEQ ID NO:6) generated by targeted homologousrecombination. Sequences derived from the vector into which theamplification product was cloned are single-underlined, chromosomalsequences not present in the donor molecule are indicated by dashedunderlining (nucleotides 32-97), sequences common to the donor and thechromosome are not underlined (nucleotides 98-394 and 402-417), andsequences unique to the donor are double-underlined (nucleotides395-401). Lower-case letters represent sequences that differ between thechromosome and the donor.

FIG. 7 shows the nucleotide sequence of a portion of the human IL2Rγgene comprising the 3′ end of the second intron and the 5′ end of thirdexon (SEQ ID NO:7) and the amino acid sequence encoded by the displayedportion of the third exon (SEQ ID NO:8). Target sequences for the secondpair of IL2Rγ-specific ZFPs are underlined. See Example 2 for details.

FIG. 8 shows a schematic diagram of a plasmid encoding a ZFP-FokI fusionfor targeted cleavage of IL2Rγ gene.

FIG. 9 A-D show a schematic diagram of the IL2Rγ gene. FIG. 9A shows aschematic of a portion of the human X chromosome which includes theIL2Rγ gene. FIG. 9B shows a schematic of a portion of the IL2Rγ geneincluding a portion of the second intron, the third exon and a portionof the third intron. Locations of sequences homologous to the initialamplification primers and to the chromosome-specific primer (see Table5) are also provided. FIG. 9C shows the nucleotide sequence of the humanX chromosome in the region of the third exon of the IL2Rγ gene (SEQ IDNO: 9), the encoded amino acid sequence (SEQ ID NO: 10), and the targetsites for the first pair of IL2Rγ-specific zinc finger proteins. FIG. 9Dshows the sequence of the corresponding region of the donor molecule(SEQ ID NO: 11), with differences between donor and chromosomalsequences underlined. Sequences contained in the donor-specificamplification primer (Table 5) are indicated by double overlining.

FIG. 10 shows a schematic diagram of the IL2Rγ donor construct.

FIG. 11 shows PCR analysis of DNA from transfected K652 cells. Fromleft, the lanes show results from cells transfected with two plasmids,each of which encodes one of a pair of IL2Rγ-specific ZFP-FokI fusionproteins (ZFPs only, lane 1), cells transfected with two concentrationsof the IL2Rγ donor plasmid (donor only, lanes 2 and 3), and cellstransfected with the two ZFP-encoding plasmids and the donor plasmid(ZFPs+donor, lanes 4-7). Each of the two pairs of IL2Rγ-specificZFP-FokI fusions were used (identified as “pair 1” and “pair 2”) and useof both pairs resulted in production of the diagnostic amplificationproduct (labeled “expected chimeric product” in the Figure). See Example2 for details.

FIG. 12 shows the nucleotide sequence of an amplification productderived from a mutated IL2Rγ gene (SEQ ID NO:12) generated by targetedhomologous recombination. Sequences derived from the vector into whichthe amplification product was cloned are single-underlined, chromosomalsequences not present in the donor molecule are indicated by dashedunderlining (nucleotides 460-552), sequences common to the donor and thechromosome are not underlined (nucleotides 32-42 and 59-459), and astretch of sequence containing nucleotides which distinguish donorsequences from chromosomal sequences is double-underlined (nucleotides44-58). Lower-case letters represent nucleotides whose sequence differsbetween the chromosome and the donor.

FIG. 13 shows the nucleotide sequence of a portion of the humanbeta-globin gene encoding segments of the core promoter, the first twoexons and the first intron (SEQ ID NO:13). A missense mutation changingan A (in boldface and underlined) at position 5212541 on Chromosome 11(BLAT, UCSC Genome Bioinformatics site) to a T results in sickle cellanemia. A first zinc finger/FokI fusion protein was designed such thatthe primary contacts were with the underlined 12-nucleotide sequenceAAGGTGAACGTG (nucleotides 305-316 of SEQ ID NO:13), and a second zincfinger/FokI fusion protein was designed such that the primary contactswere with the complement of the underlined 12-nucleotide sequenceCCGTTACTGCCC (nucleotides 325-336 of SEQ ID NO:13).

FIG. 14 is a schematic diagram of a plasmid encoding ZFP-FokI fusion fortargeted cleavage of the human beta globin gene.

FIG. 15 is a schematic diagram of the cloned human beta globin geneshowing the upstream region, first and second exons, first intron andprimer binding sites.

FIG. 16 is a schematic diagram of the beta globin donor construct,pCR4-TOPO-HBBdonor.

FIG. 17 shows PCR analysis of DNA from cells transfected with two pairsof β-globin-specific ZFP nucleases and a beta globin donor plasmid. Thepanel on the left is a loading control in which the initial amp 1 andinitial amp 2 primers (Table 7) were used for amplification. In theexperiment shown in the right panel, the “chromosome-specific and“donor-specific” primers (Table 7) were used for amplification. Theleftmost lane in each panel contains molecular weight markers and thenext lane shows amplification products obtained from mock-transfectedcells. Remaining lanes, from left to right, show amplification productfrom cells transfected with: a GFP-encoding plasmid, 100 ng of eachZFP/FokI-encoding plasmid, 200 ng of each ZFP/FokI-encoding plasmid, 200ng donor plasmid, 600 ng donor plasmid, 200 ng donor plasmid+100 ng ofeach ZFP/FokI-encoding plasmid, and 600 ng donor plasmid+200 ng of eachZFP/FokI-encoding plasmid.

FIG. 18 shows the nucleotide sequence of an amplification productderived from a mutated beta-globin gene (SEQ ID NO:14) generated bytargeted homologous recombination. Chromosomal sequences not present inthe donor molecule are indicated by dashed underlining (nucleotides1-72), sequences common to the donor and the chromosome are notunderlined (nucleotides 73-376), and a stretch of sequence containingnucleotides which distinguish donor sequences from chromosomal sequencesis double-underlined (nucleotides 377-408). Lower-case letters representnucleotides whose sequence differs between the chromosome and the donor.

FIG. 19 shows the nucleotide sequence of a portion of the fifth exon ofthe Interleukin-2 receptor gamma chain (IL-2Rγ) gene (SEQ ID NO:15).Also shown (underlined) are the target sequences for the 5-8 and 5-10ZFP/FokI fusion proteins. See Example 5 for details.

FIG. 20 shows the amino acid sequence of the 5-8 ZFP/FokI fusiontargeted to exon 5 of the human IL-2Rγ gene (SEQ ID NO:16). Amino acidresidues 1-17 contain a nuclear localization sequence (NLS, underlined);residues 18-130 contain the ZFP portion, with the recognition regions ofthe component zinc fingers shown in boldface; the ZFP-FokI linker (ZClinker, underlined) extends from residues 131 to 140 and the FokIcleavage half-domain begins at residue 141 and extends to the end of theprotein at residue 336. The residue that was altered to generate theQ486E mutation is shown underlined and in boldface.

FIG. 21 shows the amino acid sequence of the 5-10 ZFP/FokI fusiontargeted to exon 5 of the human IL-2Rγ gene (SEQ ID NO:17). Amino acidresidues 1-17 contain a nuclear localization sequence (NLS, underlined);residues 18-133 contain the ZFP portion, with the recognition regions ofthe component zinc fingers shown in boldface; the ZFP-FokI linker (ZClinker, underlined) extends from residues 134 to 143 and the FokIcleavage half-domain begins at residue 144 and extends to the end of theprotein at residue 339. The residue that was altered to generate theE490K mutation is shown underlined and in boldface.

FIG. 22 shows the nucleotide sequence of the enhanced Green FluorescentProtein gene (SEQ ID NO:18) derived from the Aequorea victoria GFP gene(Tsien (1998) Ann. Rev. Biochem. 67:509-544). The ATG initiation codon,as well as the region which was mutagenized, are underlined.

FIG. 23 shows the nucleotide sequence of a mutant defective eGFP gene(SEQ ID NO:19). Binding sites for ZFP-nucleases are underlined and theregion between the binding sites corresponds to the region that wasmodified.

FIG. 24 shows the structures of plasmids encoding Zinc Finger Nucleasestargeted to the eGFP gene.

FIG. 25 shows an autoradiogram of a 10% acrylamide gel used to analyzetargeted DNA cleavage of a mutant eGFP gene by zinc fingerendonucleases. See Example 8 for details.

FIG. 26 shows the structure of plasmid pcDNA4/TO/GFPmut (see Example 9).

FIG. 27 shows levels of eGFPmut mRNA, normalized to GAPDH mRNA, invarious cell lines obtained from transfection of human HEK293 cells.Light bars show levels in untreated cells; dark bars show levels in cellthat had been treated with 2 ng/ml doxycycline. See Example 9 fordetails.

FIG. 28 shows the structure of plasmid pCR(R)4-TOPO-GFPdonor5. SeeExample 10 for details.

FIG. 29 shows the nucleotide sequence of the eGFP insert inpCR(R)4-TOPO-GFPdonor5 (SEQ ID NO:20). The insert contains sequencesencoding a portion of a non-modified enhanced Green Fluorescent Protein,lacking an initiation codon. See Example 10 for details.

FIG. 30 shows a FACS trace of T18 cells transfected with plasmidsencoding two ZFP nucleases and a plasmid encoding a donor sequence, thatwere arrested in the G2 phase of the cell cycle 24 hourspost-transfection with 100 ng/ml nocodazole for 48 hours. The medium wasreplaced and the cells were allowed to recover for an additional 48hours, and gene correction was measured by FACS analysis. See Example 11for details.

FIG. 31 shows a FACS trace of T18 cells transfected with plasmidsencoding two ZFP nucleases and a plasmid encoding a donor sequence, thatwere arrested in the G2 phase of the cell cycle 24 hourspost-transfection with 0.2 uM vinblastine for 48 hours. The medium wasreplaced and the cells were allowed to recover for an additional 48hours, and gene correction was measured by FACS analysis. See Example 11for details.

FIG. 32 shows the nucleotide sequence of a 1,527 nucleotide eGFP insertin pCR(R)4-TOPO (SEQ ID NO:21). The sequence encodes a non-modifiedenhanced Green Fluorescent Protein lacking an initiation codon. SeeExample 13 for details.

FIG. 33 shows a schematic diagram of an assay used to measure thefrequency of editing of the endogenous human IL-2Rγ gene. See Example 14for details.

FIG. 34 shows autoradiograms of acrylamide gels used in an assay tomeasure the frequency of editing of an endogenous cellular gene bytargeted cleavage and homologous recombination. The lane labeled “GFP”shows assay results from a control in which cells were transfected withan eGFP-encoding vector; the lane labeled “ZFPs only” shows results fromanother control experiment in which cells were transfected with the twoZFP/nuclease-encoding plasmids (50 ng of each) but not with a donorsequence. Lanes labeled “donor only” show results from a controlexperiment in which cells were transfected with 1 ug of donor plasmidbut not with the ZFP/nuclease-encoding plasmids. In the experimentallanes, 50Z refers to cells transfected with 50 ng of each ZFP/nucleaseexpression plasmid, 100Z refers to cells transfected with 100 ng of eachZFP/nuclease expression plasmid, 0.5D refers to cells transfected with0.5 μg of the donor plasmid, and 1D refers to cells transfected with 1.0μg of the donor plasmid. “+” refers to cells that were exposed to 0.2 μMvinblastine; “−” refer to cells that were not exposed to vinblastine.“wt” refers to the fragment obtained after BsrBI digestion ofamplification products obtained from chromosomes containing thewild-type chromosomal IL-2Rγ gene; “rflp” refers to the two fragments(of approximately equal molecular weight) obtained after BsrBI digestionof amplification products obtained from chromosomes containing sequencesfrom the donor plasmid which had integrated by homologous recombination.

FIG. 35 shows an autoradiographic image of a four-hour exposure of a gelused in an assay to measure targeted recombination at the human IL-2Rγlocus in K562 cells. “wt” identifies a band that is diagnostic forchromosomal DNA containing the native K562 IL-2Rγ sequence; “rflp”identifies a doublet diagnostic for chromosomal DNA containing thealtered IL-2Rγ sequence present in the donor DNA molecule. The symbol“+” above a lane indicates that cells were treated with 0.2 uMvinblastine; the symbol “−” indicates that cells were not treated withvinblastine. The numbers in the “ZFP+donor” lanes indicate thepercentage of total chromosomal DNA containing sequence originallypresent in the donor DNA molecule, calculated using the “peak finder,automatic baseline” function of Molecular Dynamics' ImageQuant v. 5.1software as described in Ch. 8 of the manufacturer's manual (MolecularDynamics ImageQuant User's Guide; part 218-415). “Untr” indicatesuntransfected cells. See Example 15 for additional details.

FIG. 36 shows an autoradiographic image of a four-hour exposure of a gelused in an assay to measure targeted recombination at the human IL-2Rγlocus in K562 cells. “wt” identifies a band that is diagnostic forchromosomal DNA containing the native K562 IL-2Rγ sequence; “rflp”identifies a band that is diagnostic for chromosomal DNA containing thealtered IL-2Rγ sequence present in the donor DNA molecule. The symbol“+” above a lane indicates that cells were treated with 0.2 uMvinblastine; the symbol “−” indicates that cells were not treated withvinblastine. The numbers beneath the “ZFP+donor” lanes indicate thepercentage of total chromosomal DNA containing sequence originallypresent in the donor DNA molecule, calculated as described in Example35. See Example 15 for additional details.

FIG. 37 shows an autoradiogram of a four-hour exposure of a DNA blotprobed with a fragment specific to the human IL-2Rγ gene. The arrow tothe right of the image indicates the position of a band corresponding togenomic DNA whose sequence has been altered by homologous recombination.The symbol “+” above a lane indicates that cells were treated with 0.2uM vinblastine; the symbol “−” indicates that cells were not treatedwith vinblastine. The numbers beneath the “ZFP+donor” lanes indicate thepercentage of total chromosomal DNA containing sequence originallypresent in the donor DNA molecule, calculated as described in Example35. See Example 15 for additional details.

FIG. 38 shows autoradiographic images of gels used in an assay tomeasure targeted recombination at the human IL-2Rγ locus in CD34⁺ humanbone marrow cells. The left panel shows a reference standard in whichthe stated percentage of normal human genomic DNA (containing a MaeIIsite), was added to genomic DNA from Jurkat cells (lacking a MaeIIsite), the mixture was amplified by PCR to generate a radiolabelledamplification product, and the amplification product was digested withMaeII. “wt” identifies a band representing undigested DNA, and “rflp”identifies a band resulting from MaeII digestion.

The right panel shows results of an experiment in which CD34⁺ cells weretransfected with donor DNA containing a BsrBI site and plasmids encodingzinc finger-FokI fusion endonucleases. The relevant genomic region wasthen amplified and labeled, and the labeled amplification product wasdigested with BsrBI. “GFP” indicates control cells that were transfectedwith a GFP-encoding plasmid; “Donor only” indicates control cells thatwere transfected only with donor DNA, and “ZFP+Donor” indicates cellsthat were transfected with donor DNA and with plasmids encoding the zincfinger/FokI nucleases. “wt” identifies a band that is diagnostic forchromosomal DNA containing the native IL-2Rγ sequence; “rflp” identifiesa band that is diagnostic for chromosomal DNA containing the alteredIL-2Rγ sequence present in the donor DNA molecule. The rightmost lanecontains DNA size markers. See Example 16 for additional details.

FIG. 39 shows an image of an immunoblot used to test for Ku70 proteinlevels in cells transfected with Ku70-targeted siRNA. The T7 cell line(Example 9, FIG. 27) was transfected with two concentrations each ofsiRNA from two different siRNA pools (see Example 18). Lane 1: 70 ng ofsiRNA pool D; Lane 2: 140 ng of siRNA pool D; Lane 3: 70 ng of siRNApool E; Lane 4: 140 ng of siRNA pool E. “Ku70” indicates the bandrepresenting the Ku70 protein; “TFIIB” indicates a band representing theTFIIB transcription factor, used as a control.

FIG. 40 shows the amino acid sequences of four zinc finger domainstargeted to the human β-globin gene: sca-29b (SEQ ID NO:22); sca-36a(SEQ ID NO:23); sca-36b (SEQ ID NO:24) and sca-36c (SEQ ID NO:25). Thetarget site for the sca-29b domain is on one DNA strand, and the targetsites for the sca-36a, sca-36b and sca-36c domains are on the oppositestrand. See Example 20.

FIG. 41 shows results of an in vitro assay, in which differentcombinations of zinc finger/FokI fusion nucleases (ZFNs) were tested forsequence-specific DNA cleavage. The lane labeled “U” shows a sample ofthe DNA template. The next four lanes show results of incubation of theDNA template with each of four β-globin-targeted ZFNs (see Example 20for characterization of these ZFNs). The rightmost three lanes showresults of incubation of template DNA with the sca-29b ZFN and one ofthe sca-36a, sca-36b or sca-36c ZFNs (all of which are targeted to thestrand opposite that to which sca-29b is targeted).

FIG. 42 shows levels of eGFP mRNA in T18 cells (bars) as a function ofdoxycycline concentration (provided on the abscissa). The number aboveeach bar represents the percentage correction of the eGFP mutation, incells transfected with donor DNA and plasmids encoding eGFP-targetedzinc finger nucleases, as a function of doxycycline concentration.

DETAILED DESCRIPTION

Disclosed herein are compositions and methods useful for targetedcleavage of cellular chromatin and for targeted alteration of a cellularnucleotide sequence, e.g., by targeted cleavage followed bynon-homologous end joining or by targeted cleavage followed byhomologous recombination between an exogenous polynucleotide (comprisingone or more regions of homology with the cellular nucleotide sequence)and a genomic sequence. Genomic sequences include those present inchromosomes, episomes, organellar genomes (e.g., mitochondria,chloroplasts), artificial chromosomes and any other type of nucleic acidpresent in a cell such as, for example, amplified sequences, doubleminute chromosomes and the genomes of endogenous or infecting bacteriaand viruses. Genomic sequences can be normal (i.e., wild-type) ormutant; mutant sequences can comprise, for example, insertions,deletions, translocations, rearrangements, and/or point mutations. Agenomic sequence can also comprise one of a number of different alleles.

Compositions useful for targeted cleavage and recombination includefusion proteins comprising a cleavage domain (or a cleavage half-domain)and a zinc finger binding domain, polynucleotides encoding theseproteins and combinations of polypeptides and polypeptide-encodingpolynucleotides. A zinc finger binding domain can comprise one or morezinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers), andcan be engineered to bind to any genomic sequence. Thus, by identifyinga target genomic region of interest at which cleavage or recombinationis desired, one can, according to the methods disclosed herein,construct one or more fusion proteins comprising a cleavage domain (orcleavage half-domain) and a zinc finger domain engineered to recognize atarget sequence in said genomic region. The presence of such a fusionprotein (or proteins) in a cell will result in binding of the fusionprotein(s) to its (their) binding site(s) and cleavage within or nearsaid genomic region. Moreover, if an exogenous polynucleotide homologousto the genomic region is also present in such a cell, homologousrecombination occurs at a high rate between the genomic region and theexogenous polynucleotide.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

DEFINITIONS

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

Zinc finger binding domains can be “engineered” to bind to apredetermined nucleotide sequence. Non-limiting examples of methods forengineering zinc finger proteins are design and selection. A designedzinc finger protein is a protein not occurring in nature whosedesign/composition results principally from rational criteria. Rationalcriteria for design include application of substitution rules andcomputerized algorithms for processing information in a database storinginformation of existing ZFP designs and binding data. See, for example,U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

A “selected” zinc finger protein is a protein not found in nature whoseproduction results primarily from an empirical process such as phagedisplay, interaction trap or hybrid selection. See e.g., U.S. Pat. No.5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988; U.S. Pat.No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO 96/06166; WO98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197 and WO02/099084.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

A “homologous, non-identical sequence” refers to a first sequence whichshares a degree of sequence identity with a second sequence, but whosesequence is not identical to that of the second sequence. For example, apolynucleotide comprising the wild-type sequence of a mutant gene ishomologous and non-identical to the sequence of the mutant gene. Incertain embodiments, the degree of homology between the two sequences issufficient to allow homologous recombination therebetween, utilizingnormal cellular mechanisms. Two homologous non-identical sequences canbe any length and their degree of non-homology can be as small as asingle nucleotide (e.g., for correction of a genomic point mutation bytargeted homologous recombination) or as large as 10 or more kilobases(e.g., for insertion of a gene at a predetermined ectopic site in achromosome). Two polynucleotides comprising the homologous non-identicalsequences need not be the same length. For example, an exogenouspolynucleotide (i.e., donor polynucleotide) of between 20 and 10,000nucleotides or nucleotide pairs can be used:

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100. An approximatealignment for nucleic acid sequences is provided by the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics2:482-489 (1981). This algorithm can be applied to amino acid sequencesby using the scoring matrix developed by Dayhoff, Atlas of ProteinSequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, NationalBiomedical Research Foundation, Washington, D.C., USA, and normalized byGribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplaryimplementation of this algorithm to determine percent identity of asequence is provided by the Genetics Computer Group (Madison, Wis.) inthe “BestFit” utility application. The default parameters for thismethod are described in the Wisconsin Sequence Analysis Package ProgramManual, Version 8 (1995) (available from Genetics Computer Group,Madison, Wis.). A preferred method of establishing percent identity inthe context of the present disclosure is to use the MPSRCH package ofprograms copyrighted by the University of Edinburgh, developed by JohnF. Collins and Shane S. Sturrok, and distributed by IntelliGenetics,Inc. (Mountain View, Calif.). From this suite of packages theSmith-Waterman algorithm can be employed where default parameters areused for the scoring table (for example, gap open penalty of 12, gapextension penalty of one, and a gap of six). From the data generated the“Match” value reflects sequence identity. Other suitable programs forcalculating the percent identity or similarity between sequences aregenerally known in the art, for example, another alignment program isBLAST, used with default parameters. For example, BLASTN and BLASTP canbe used using the following default parameters: genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found online.With respect to sequences described herein, the range of desired degreesof sequence identity is approximately 80% to 100% and any integer valuetherebetween. Typically the percent identities between sequences are atleast 70-75%, preferably 80-82%, more preferably 85-90%, even morepreferably 92%, still more preferably 95%, and most preferably 98%sequence identity.

Alternatively, the degree of sequence similarity between polynucleotidescan be determined by hybridization of polynucleotides under conditionsthat allow formation of stable duplexes between homologous regions,followed by digestion with single-stranded-specific nuclease(s), andsize determination of the digested fragments. Two nucleic acid, or twopolypeptide sequences are substantially homologous to each other whenthe sequences exhibit at least about 70%-75%, preferably 80%-82%, morepreferably 85%-90%, even more preferably 92%, still more preferably 95%,and most preferably 98% sequence identity over a defined length of themolecules, as determined using the methods above. As used herein,substantially homologous also refers to sequences showing completeidentity to a specified DNA or polypeptide sequence. DNA sequences thatare substantially homologous can be identified in a Southernhybridization experiment under, for example, stringent conditions, asdefined for that particular system. Defining appropriate hybridizationconditions is within the skill of the art. See, e.g., Sambrook et al.,supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D.Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

Selective hybridization of two nucleic acid fragments can be determinedas follows. The degree of sequence identity between two nucleic acidmolecules affects the efficiency and strength of hybridization eventsbetween such molecules. A partially identical nucleic acid sequence willat least partially inhibit the hybridization of a completely identicalsequence to a target molecule. Inhibition of hybridization of thecompletely identical sequence can be assessed using hybridization assaysthat are well known in the art (e.g., Southern (DNA) blot, Northern(RNA) blot, solution hybridization, or the like, see Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.). Such assays can be conducted using varying degreesof selectivity, for example, using conditions varying from low to highstringency. If conditions of low stringency are employed, the absence ofnon-specific binding can be assessed using a secondary probe that lackseven a partial degree of sequence identity (for example, a probe havingless than about 30% sequence identity with the target molecule), suchthat, in the absence of non-specific binding events, the secondary probewill not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a reference nucleic acidsequence, and then by selection of appropriate conditions the probe andthe reference sequence selectively hybridize, or bind, to each other toform a duplex molecule. A nucleic acid molecule that is capable ofhybridizing selectively to a reference sequence under moderatelystringent hybridization conditions typically hybridizes under conditionsthat allow detection of a target nucleic acid sequence of at least about10-14 nucleotides in length having at least approximately 70% sequenceidentity with the sequence of the selected nucleic acid probe. Stringenthybridization conditions typically allow detection of target nucleicacid sequences of at least about 10-14 nucleotides in length having asequence identity of greater than about 90-95% with the sequence of theselected nucleic acid probe. Hybridization conditions useful forprobe/reference sequence hybridization, where the probe and referencesequence have a specific degree of sequence identity, can be determinedas is known in the art (see, for example, Nucleic Acid Hybridization: APractical Approach, editors B. D. Hames and S. J. Higgins, (1985)Oxford; Washington, D.C.; IRL Press).

Conditions for hybridization are well-known to those of skill in theart. Hybridization stringency refers to the degree to whichhybridization conditions disfavor the formation of hybrids containingmismatched nucleotides, with higher stringency correlated with a lowertolerance for mismatched hybrids. Factors that affect the stringency ofhybridization are well-known to those of skill in the art and include,but are not limited to, temperature, pH, ionic strength, andconcentration of organic solvents such as, for example, formamide anddimethylsulfoxide. As is known to those of skill in the art,hybridization stringency is increased by higher temperatures, lowerionic strength and lower solvent concentrations.

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of the sequences, base composition of thevarious sequences, concentrations of salts and other hybridizationsolution components, the presence or absence of blocking agents in thehybridization solutions (e.g., dextran sulfate, and polyethyleneglycol), hybridization reaction temperature and time parameters, as wellas, varying wash conditions. The selection of a particular set ofhybridization conditions is selected following standard methods in theart (see, for example, Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, (1989) Cold Spring Harbor, N.Y.).

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells. This process requires nucleotide sequence homology,uses a “donor” molecule to template repair of a “target” molecule (i.e.,the one that experienced the double-strand break), and is variouslyknown as “non-crossover gene conversion” or “short tract geneconversion,” because it leads to the transfer of genetic informationfrom the donor to the target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or “synthesis-dependent strand annealing,” in which the donor isused to resynthesize genetic information that will become part of thetarget, and/or related processes. Such specialized HR often results inan alteration of the sequence of the target molecule such that part orall of the sequence of the donor polynucleotide is incorporated into thetarget polynucleotide.

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage domain” comprises one or more polypeptide sequences whichpossesses catalytic activity for DNA cleavage. A cleavage domain can becontained in a single polypeptide chain or cleavage activity can resultfrom the association of two (or more) polypeptides.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity).

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

An “accessible region” is a site in cellular chromatin in which a targetsite present in the nucleic acid can be bound by an exogenous moleculewhich recognizes the target site. Without wishing to be bound by anyparticular theory, it is believed that an accessible region is one thatis not packaged into a nucleosomal structure. The distinct structure ofan accessible region can often be detected by its sensitivity tochemical and enzymatic probes, for example, nucleases.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist. For example, thesequence 5′-GAATTC-3′ is a target site for the Eco RI restrictionendonuclease.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPDNA-binding domain and a cleavage domain) and fusion nucleic acids (forexample, a nucleic acid encoding the fusion protein described supra).Examples of the second type of fusion molecule include, but are notlimited to, a fusion between a triplex-forming nucleic acid and apolypeptide, and a fusion between a minor groove binder and a nucleicacid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of a mRNA. Gene products also include RNAs whichare modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression.

“Eucaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFPDNA-binding domain is fused to a cleavage domain, the ZFP DNA-bindingdomain and the cleavage domain are in operative linkage if, in thefusion polypeptide, the ZFP DNA-binding domain portion is able to bindits target site and/or its binding site, while the cleavage domain isable to cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

Target Sites

The disclosed methods and compositions include fusion proteinscomprising a cleavage domain (or a cleavage half-domain) and a zincfinger domain, in which the zinc finger domain, by binding to a sequencein cellular chromatin (e.g., a target site or a binding site), directsthe activity of the cleavage domain (or cleavage half-domain) to thevicinity of the sequence and, hence, induces cleavage in the vicinity ofthe target sequence. As set forth elsewhere in this disclosure, a zincfinger domain can be engineered to bind to virtually any desiredsequence. Accordingly, after identifying a region of interest containinga sequence at which cleavage or recombination is desired, one or morezinc finger binding domains can be engineered to bind to one or moresequences in the region of interest. Expression of a fusion proteincomprising a zinc finger binding domain and a cleavage domain (or of twofusion proteins, each comprising a zinc finger binding domain and acleavage half-domain), in a cell, effects cleavage in the region ofinterest.

Selection of a sequence in cellular chromatin for binding by a zincfinger domain (e.g., a target site) can be accomplished, for example,according to the methods disclosed in co-owned U.S. Pat. No. 6,453,242(Sep. 17, 2002), which also discloses methods for designing ZFPs to bindto a selected sequence. It will be clear to those skilled in the artthat simple visual inspection of a nucleotide sequence can also be usedfor selection of a target site. Accordingly, any means for target siteselection can be used in the claimed methods.

Target sites are generally composed of a plurality of adjacent targetsubsites. A target subsite refers to the sequence (usually either anucleotide triplet, or a nucleotide quadruplet that can overlap by onenucleotide with an adjacent quadruplet) bound by an individual zincfinger. See, for example, WO 02/077227. If the strand with which a zincfinger protein makes most contacts is designated the target strand“primary recognition strand,” or “primary contact strand,” some zincfinger proteins bind to a three base triplet in the target strand and afourth base on the non-target strand. A target site generally has alength of at least 9 nucleotides and, accordingly, is bound by a zincfinger binding domain comprising at least three zinc fingers. Howeverbinding of, for example, a 4-finger binding domain to a 12-nucleotidetarget site, a 5-finger binding domain to a 15-nucleotide target site ora 6-finger binding domain to an 18-nucleotide target site, is alsopossible. As will be apparent, binding of larger binding domains (e.g.,7-, 8-, 9-finger and more) to longer target sites is also possible.

It is not necessary for a target site to be a multiple of threenucleotides. For example, in cases in which cross-strand interactionsoccur (see, e.g., U.S. Pat. No. 6,453,242 and WO 02/077227), one or moreof the individual zinc fingers of a multi-finger binding domain can bindto overlapping quadruplet subsites. As a result, a three-finger proteincan bind a 10-nucleotide sequence, wherein the tenth nucleotide is partof a quadruplet bound by a terminal finger, a four-finger protein canbind a 13-nucleotide sequence, wherein the thirteenth nucleotide is partof a quadruplet bound by a terminal finger, etc.

The length and nature of amino acid linker sequences between individualzinc fingers in a multi-finger binding domain also affects binding to atarget sequence. For example, the presence of a so-called “non-canonicallinker,” “long linker” or “structured linker” between adjacent zincfingers in a multi-finger binding domain can allow those fingers to bindsubsites which are not immediately adjacent. Non-limiting examples ofsuch linkers are described, for example, in U.S. Pat. No. 6,479,626 andWO 01/53480. Accordingly, one or more subsites, in a target site for azinc finger binding domain, can be separated from each other by 1, 2, 3,4, 5 or more nucleotides. To provide but one example, a four-fingerbinding domain can bind to a 13-nucleotide target site comprising, insequence, two contiguous 3-nucleotide subsites, an interveningnucleotide, and two contiguous triplet subsites.

Distance between sequences (e.g., target sites) refers to the number ofnucleotides or nucleotide pairs intervening between two sequences, asmeasured from the edges of the sequences nearest each other.

In certain embodiments in which cleavage depends on the binding of twozinc finger domain/cleavage half-domain fusion molecules to separatetarget sites, the two target sites can be on opposite DNA strands. Inother embodiments, both target sites are on the same DNA strand.

Zinc Finger Binding Domains

A zinc finger binding domain comprises one or more zinc fingers. Milleret al. (1985) EMBO J. 4:1609-1614; Rhodes (1993) Scientific AmericanFeb.:56-65; U.S. Pat. No. 6,453,242. Typically, a single zinc fingerdomain is about 30 amino acids in length. Structural studies havedemonstrated that each zinc finger domain (motif) contains two betasheets (held in a beta turn which contains the two invariant cysteineresidues) and an alpha helix (containing the two invariant histidineresidues), which are held in a particular conformation throughcoordination of a zinc atom by the two cysteines and the two histidines.

Zinc fingers include both canonical C₂H₂ zinc fingers (i.e., those inwhich the zinc ion is coordinated by two cysteine and two histidineresidues) and non-canonical zinc fingers such as, for example, C₃H zincfingers (those in which the zinc ion is coordinated by three cysteineresidues and one histidine residue) and C₄ zinc fingers (those in whichthe zinc ion is coordinated by four cysteine residues). See also WO02/057293.

Zinc finger binding domains can be engineered to bind to a sequence ofchoice. See, for example, Beerli et al. (2002) Nature Biotechnol.20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan etal. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr.Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct.Biol. 10:411-416. An engineered zinc finger binding domain can have anovel binding specificity, compared to a naturally-occurring zinc fingerprotein. Engineering methods include, but are not limited to, rationaldesign and various types of selection. Rational design includes, forexample, using databases comprising triplet (or quadruplet) nucleotidesequences and individual zinc finger amino acid sequences, in which eachtriplet or quadruplet nucleotide sequence is associated with one or moreamino acid sequences of zinc fingers which bind the particular tripletor quadruplet sequence. See, for example, co-owned U.S. Pat. Nos.6,453,242 and 6,534,261.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237.

Enhancement of binding specificity for zinc finger binding domains hasbeen described, for example, in co-owned WO 02/077227.

Since an individual zinc finger binds to a three-nucleotide (i.e.,triplet) sequence (or a four-nucleotide sequence which can overlap, byone nucleotide, with the four-nucleotide binding site of an adjacentzinc finger), the length of a sequence to which a zinc finger bindingdomain is engineered to bind (e.g., a target sequence) will determinethe number of zinc fingers in an engineered zinc finger binding domain.For example, for ZFPs in which the finger motifs do not bind tooverlapping subsites, a six-nucleotide target sequence is bound by atwo-finger binding domain; a nine-nucleotide target sequence is bound bya three-finger binding domain, etc. As noted herein, binding sites forindividual zinc fingers (i.e., subsites) in a target site need not becontiguous, but can be separated by one or several nucleotides,depending on the length and nature of the amino acids sequences betweenthe zinc fingers (i.e., the inter-finger linkers) in a multi-fingerbinding domain.

In a multi-finger zinc finger binding domain, adjacent zinc fingers canbe separated by amino acid linker sequences of approximately 5 aminoacids (so-called “canonical” inter-finger linkers) or, alternatively, byone or more non-canonical linkers. See, e.g., co-owned U.S. Pat. Nos.6,453,242 and 6,534,261. For engineered zinc finger binding domainscomprising more than three fingers, insertion of longer(“non-canonical”) inter-finger linkers between certain of the zincfingers may be preferred as it may increase the affinity and/orspecificity of binding by the binding domain. See, for example, U.S.Pat. No. 6,479,626 and WO 01/53480. Accordingly, multi-finger zincfinger binding domains can also be characterized with respect to thepresence and location of non-canonical inter-finger linkers. Forexample, a six-finger zinc finger binding domain comprising threefingers (joined by two canonical inter-finger linkers), a long linkerand three additional fingers (joined by two canonical inter-fingerlinkers) is denoted a 2×3 configuration. Similarly, a binding domaincomprising two fingers (with a canonical linker therebetween), a longlinker and two additional fingers (joined by a canonical linker) isdenoted a 2×2 protein. A protein comprising three two-finger units (ineach of which the two fingers are joined by a canonical linker), and inwhich each two-finger unit is joined to the adjacent two finger unit bya long linker, is referred to as a 3×2 protein.

The presence of a long or non-canonical inter-finger linker between twoadjacent zinc fingers in a multi-finger binding domain often allows thetwo fingers to bind to subsites which are not immediately contiguous inthe target sequence. Accordingly, there can be gaps of one or morenucleotides between subsites in a target site; i.e., a target site cancontain one or more nucleotides that are not contacted by a zinc finger.For example, a 2×2 zinc finger binding domain can bind to twosix-nucleotide sequences separated by one nucleotide, i.e., it binds toa 13-nucleotide target site. See also Moore et al. (2001a) Proc. Natl.Acad. Sci. USA 98:1432-1436; Moore et al. (2001b) Proc. Natl. Acad. Sci.USA 98:1437-1441 and WO 01/53480.

As mentioned previously, a target subsite is a three- or four-nucleotidesequence that is bound by a single zinc finger. For certain purposes, atwo-finger unit is denoted a binding module. A binding module can beobtained by, for example, selecting for two adjacent fingers in thecontext of a multi-finger protein (generally three fingers) which bind aparticular six-nucleotide target sequence. Alternatively, modules can beconstructed by assembly of individual zinc fingers. See also WO 98/53057and WO 01/53480.

Cleavage Domains

The cleavage domain portion of the fusion proteins disclosed herein canbe obtained from any endo- or exonuclease. Exemplary endonucleases fromwhich a cleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., S1 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional fragments thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain (e.g., fusion proteins comprising azinc finger binding domain and a cleavage half-domain) can be derivedfrom any nuclease or portion thereof, as set forth above, that requiresdimerization for cleavage activity. In general, two fusion proteins arerequired for cleavage if the fusion proteins comprise cleavagehalf-domains. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins places the cleavage half-domains in aspatial orientation to each other that allows the cleavage half-domainsto form a functional cleavage domain, e.g., by dimerizing. Thus, incertain embodiments, the near edges of the target sites are separated by5-8 nucleotides or by 15-18 nucleotides. However any integral number ofnucleotides or nucleotide pairs can intervene between two target sites(e.g., from 2 to 50 nucleotides or more). In general, the point ofcleavage lies between the target sites.

In general, if two fusion proteins are used, each comprising a cleavagehalf-domain, the primary contact strand for the zinc finger portion ofeach fusion protein will be on a different DNA strands and in oppositeorientation. That is, for a pair of ZFP/cleavage half-domain fusions,the target sequences are on opposite strands and the two proteins bindin opposite orientations.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme FokI catalyzes double-strandedcleavage of DNA, at 9 nucleotides from its recognition site on onestrand and 13 nucleotides from its recognition site on the other. See,for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as wellas Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al.(1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc.Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise thecleavage domain (or cleavage half-domain) from at least one Type IISrestriction enzyme and one or more zinc finger binding domains, whichmay or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the Fok I enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-Fok Ifusions are provided elsewhere in this disclosure.

Exemplary Type IIS restriction enzymes are listed in Table 1. Additionalrestriction enzymes also contain separable binding and cleavage domains,and these are contemplated by the present disclosure. See, for example,Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

TABLE 1 Some Type IIS Restriction Enzymes Aar I Ace III Aci I Alo I BaeI Bbr7 I Bbv I Bbv II BbvC I Bcc I Bce83 I BceA I Bcef I Bcg I BciV IBfi I Bin I Bmg I Bpu10 I BsaX I Bsb I BscA I BscG I BseR I BseY I Bsi IBsm I BsmA I BsmF I Bsp24 I BspG I BspM I BspNC I Bsr I BsrB I BsrD IBstF5 I Btr I Bts I Cdi I CjeP I Drd II Eci I Eco31 I Eco57 I Eco57M IEsp3 I Fau I Fin I Fok I Gdi II Gsu I Hga I Hin4 II Hph I Ksp632 I MboII Mly I Mme I Mnl I Pfl1108 I Ple I Ppi I Psr I RleA I Sap I SfaN I SimI SspD5 I Sth132 I Sts I TspDT I TspGW I Tth111 II UbaP I Bsa I BsmB I

Zinc Finger Domain-Cleavage Domain Fusions

Methods for design and construction of fusion proteins (andpolynucleotides encoding same) are known to those of skill in the art.For example, methods for the design and construction of fusion proteincomprising zinc finger proteins (and polynucleotides encoding same) aredescribed in co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261. In certainembodiments, polynucleotides encoding such fusion proteins areconstructed. These polynucleotides can be inserted into a vector and thevector can be introduced into a cell (see below for additionaldisclosure regarding vectors and methods for introducing polynucleotidesinto cells).

In certain embodiments of the methods described herein, a fusion proteincomprises a zinc finger binding domain and a cleavage half-domain fromthe Fok I restriction enzyme, and two such fusion proteins are expressedin a cell. Expression of two fusion proteins in a cell can result fromdelivery of the two proteins to the cell; delivery of one protein andone nucleic acid encoding one of the proteins to the cell; delivery oftwo nucleic acids, each encoding one of the proteins, to the cell; or bydelivery of a single nucleic acid, encoding both proteins, to the cell.In additional embodiments, a fusion protein comprises a singlepolypeptide chain comprising two cleavage half domains and a zinc fingerbinding domain. In this case, a single fusion protein is expressed in acell and, without wishing to be bound by theory, is believed to cleaveDNA as a result of formation of an intramolecular dimer of the cleavagehalf-domains.

In general, the components of the fusion proteins (e.g, ZFP-Fok Ifusions) are arranged such that the zinc finger domain is nearest theamino terminus of the fusion protein, and the cleavage half-domain isnearest the carboxy-terminus. This mirrors the relative orientation ofthe cleavage domain in naturally-occurring dimerizing cleavage domainssuch as those derived from the Fok I enzyme, in which the DNA-bindingdomain is nearest the amino terminus and the cleavage half-domain isnearest the carboxy terminus.

In the disclosed fusion proteins, the amino acid sequence between thezinc finger binding domain (which is delimited by the N-terminal most ofthe two conserved cysteine residues and the C-terminal-most of the twoconserved histidine residues) and the cleavage domain (or half-domain)is denoted the “ZC linker.” The ZC linker is to be distinguished fromthe inter-finger linkers discussed above. For instance, in a ZFP-Fok Ifusion protein (in which the components are arranged: N terminus-zincfinger binding domain-Fok I cleavage half domain-C terminus), the ZClinker is located between the second histidine residue of theC-terminal-most zinc finger and the N-terminal-most amino acid residueof the cleavage half-domain (which is generally glutamine (Q) in thesequence QLV). The ZC linker can be any amino acid sequence. To obtainoptimal cleavage, the length of the linker and the distance between thetarget sites (binding sites) are interrelated. See, for example, Smithet al. (2000) Nucleic Acids Res. 28:3361-3369; Bibikova et al. (2001)Mol. Cell. Biol. 21:289-297, noting that their notation for linkerlength differs from that given here. For example, for ZFP-Fok I fusionshaving a ZC linker length of four amino acids (as defined herein),optimal cleavage occurs when the binding sites for the fusion proteinsare located 6 or 16 nucleotides apart (as measured from the near edge ofeach binding site).

Methods for Targeted Cleavage

The disclosed methods and compositions can be used to cleave DNA at aregion of interest in cellular chromatin (e.g., at a desired orpredetermined site in a genome, for example, in a gene, either mutant orwild-type). For such targeted DNA cleavage, a zinc finger binding domainis engineered to bind a target site at or near the predeterminedcleavage site, and a fusion protein comprising the engineered zincfinger binding domain and a cleavage domain is expressed in a cell. Uponbinding of the zinc finger portion of the fusion protein to the targetsite, the DNA is cleaved near the target site by the cleavage domain.The exact site of cleavage can depend on the length of the ZC linker.

Alternatively, two fusion proteins, each comprising a zinc fingerbinding domain and a cleavage half-domain, are expressed in a cell, andbind to target sites which are juxtaposed in such a way that afunctional cleavage domain is reconstituted and DNA is cleaved in thevicinity of the target sites. In one embodiment, cleavage occurs betweenthe target sites of the two zinc finger binding domains. One or both ofthe zinc finger binding domains can be engineered.

For targeted cleavage using a zinc finger binding domain-cleavage domainfusion polypeptide, the binding site can encompass the cleavage site, orthe near edge of the binding site can be 1, 2, 3, 4, 5, 6, 10, 25, 50 ormore nucleotides (or any integral value between 1 and 50 nucleotides)from the cleavage site. The exact location of the binding site, withrespect to the cleavage site, will depend upon the particular cleavagedomain, and the length of the ZC linker. For methods in which two fusionpolypeptides, each comprising a zinc finger binding domain and acleavage half-domain, are used, the binding sites generally straddle thecleavage site. Thus the near edge of the first binding site can be 1, 2,3, 4, 5, 6, 10, 25 or more nucleotides (or any integral value between 1and 50 nucleotides) on one side of the cleavage site, and the near edgeof the second binding site can be 1, 2, 3, 4, 5, 6, 10, 25 or morenucleotides (or any integral value between 1 and 50 nucleotides) on theother side of the cleavage site. Methods for mapping cleavage sites invitro and in vivo are known to those of skill in the art.

Thus, the methods described herein can employ an engineered zinc fingerbinding domain fused to a cleavage domain. In these cases, the bindingdomain is engineered to bind to a target sequence, at or near whichcleavage is desired. The fusion protein, or a polynucleotide encodingsame, is introduced into a cell. Once introduced into, or expressed in,the cell, the fusion protein binds to the target sequence and cleaves ator near the target sequence. The exact site of cleavage depends on thenature of the cleavage domain and/or the presence and/or nature oflinker sequences between the binding and cleavage domains. In caseswhere two fusion proteins, each comprising a cleavage half-domain, areused, the distance between the near edges of the binding sites can be 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 25 or more nucleotides (or any integralvalue between 1 and 50 nucleotides). Optimal levels of cleavage can alsodepend on both the distance between the binding sites of the two fusionproteins (See, for example, Smith et al. (2000) Nucleic Acids Res.28:3361-3369; Bibikova et al. (2001) Mol. Cell. Biol. 21:289-297) andthe length of the ZC linker in each fusion protein.

For ZFP-FokI fusion nucleases, the length of the linker between the ZFPand the FokI cleavage half-domain (i.e., the ZC linker) can influencecleavage efficiency. In one experimental system utilizing a ZFP-FokIfusion with a ZC linker of 4 amino acid residues, optimal cleavage wasobtained when the near edges of the binding sites for two ZFP-FokInucleases were separated by 6 base pairs. This particular fusionnuclease comprised the following amino acid sequence between the zincfinger portion and the nuclease half-domain:

HQRTHQNKKQLV (SEQ ID NO: 26)in which the two conserved histidines in the C-terminal portion of thezinc finger and the first three residues in the FokI cleavagehalf-domain are underlined. Accordingly, the linker sequence in thisconstruct is QNKK. Bibikova et al. (2001) Mol. Cell. Biol. 21:289-297.The present inventors have constructed a number of ZFP-FokI fusionnucleases having a variety of ZC linker lengths and sequences, andanalyzed the cleavage efficiencies of these nucleases on a series ofsubstrates having different distances between the ZFP binding sites. SeeExample 4.

In certain embodiments, the cleavage domain comprises two cleavagehalf-domains, both of which are part of a single polypeptide comprisinga binding domain, a first cleavage half-domain and a second cleavagehalf-domain. The cleavage half-domains can have the same amino acidsequence or different amino acid sequences, so long as they function tocleave the DNA.

Cleavage half-domains may also be provided in separate molecules. Forexample, two fusion polypeptides may be introduced into a cell, whereineach polypeptide comprises a binding domain and a cleavage half-domain.The cleavage half-domains can have the same amino acid sequence ordifferent amino acid sequences, so long as they function to cleave theDNA. Further, the binding domains bind to target sequences which aretypically disposed in such a way that, upon binding of the fusionpolypeptides, the two cleavage half-domains are presented in a spatialorientation to each other that allows reconstitution of a cleavagedomain (e.g., by dimerization of the half-domains), thereby positioningthe half-domains relative to each other to form a functional cleavagedomain, resulting in cleavage of cellular chromatin in a region ofinterest. Generally, cleavage by the reconstituted cleavage domainoccurs at a site located between the two target sequences. One or bothof the proteins can be engineered to bind to its target site.

The two fusion proteins can bind in the region of interest in the sameor opposite polarity, and their binding sites (i.e., target sites) canbe separated by any number of nucleotides, e.g., from 0 to 200nucleotides or any integral value therebetween. In certain embodiments,the binding sites for two fusion proteins, each comprising a zinc fingerbinding domain and a cleavage half-domain, can be located between 5 and18 nucleotides apart, for example, 5-8 nucleotides apart, or 15-18nucleotides apart, or 6 nucleotides apart, or 16 nucleotides apart, asmeasured from the edge of each binding site nearest the other bindingsite, and cleavage occurs between the binding sites.

The site at which the DNA is cleaved generally lies between the bindingsites for the two fusion proteins. Double-strand breakage of DNA oftenresults from two single-strand breaks, or “nicks,” offset by 1, 2, 3, 4,5, 6 or more nucleotides, (for example, cleavage of double-stranded DNAby native Fok I results from single-strand breaks offset by 4nucleotides). Thus, cleavage does not necessarily occur at exactlyopposite sites on each DNA strand. In addition, the structure of thefusion proteins and the distance between the target sites can influencewhether cleavage occurs adjacent a single nucleotide pair, or whethercleavage occurs at several sites. However, for many applications,including targeted recombination (see infra) cleavage within a range ofnucleotides is generally sufficient, and cleavage between particularbase pairs is not required.

As noted above, the fusion protein(s) can be introduced as polypeptidesand/or polynucleotides. For example, two polynucleotides, eachcomprising sequences encoding one of the aforementioned polypeptides,can be introduced into a cell, and when the polypeptides are expressedand each binds to its target sequence, cleavage occurs at or near thetarget sequence. Alternatively, a single polynucleotide comprisingsequences encoding both fusion polypeptides is introduced into a cell.Polynucleotides can be DNA, RNA or any modified forms or analogues orDNA and/or RNA.

To enhance cleavage specificity, additional compositions may also beemployed in the methods described herein. For example, single cleavagehalf-domains can exhibit limited double-stranded cleavage activity. Inmethods in which two fusion proteins, each containing a three-fingerzinc finger domain and a cleavage half-domain, are introduced into thecell, either protein specifies an approximately 9-nucleotide targetsite. Although the aggregate target sequence of 18 nucleotides is likelyto be unique in a mammalian genome, any given 9-nucleotide target siteoccurs, on average, approximately 23,000 times in the human genome.Thus, non-specific cleavage, due to the site-specific binding of asingle half-domain, may occur. Accordingly, the methods described hereincontemplate the use of a dominant-negative mutant of a cleavagehalf-domain such as Fok I (or a nucleic acid encoding same) that isexpressed in a cell along with the two fusion proteins. Thedominant-negative mutant is capable of dimerizing but is unable tocleave, and also blocks the cleavage activity of a half-domain to whichit is dimerized. By providing the dominant-negative mutant in molarexcess to the fusion proteins, only regions in which both fusionproteins are bound will have a high enough local concentration offunctional cleavage half-domains for dimerization and cleavage to occur.At sites where only one of the two fusion proteins are bound, itscleavage half-domain forms a dimer with the dominant negative mutanthalf-domain, and undesirable, non-specific cleavage does not occur.

Three catalytic amino acid residues in the Fok I cleavage half-domainhave been identified: Asp 450, Asp 467 and Lys 469. Bitinaite et al.(1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575. Thus, one or moremutations at one of these residues can be used to generate a dominantnegative mutation. Further, many of the catalytic amino acid residues ofother Type IIS endonucleases are known and/or can be determined, forexample, by alignment with Fok I sequences and/or by generation andtesting of mutants for catalytic activity.

Dimerization Domain Mutations in the Cleavage Half-Domain

Methods for targeted cleavage which involve the use of fusions between aZFP and a cleavage half-domain (such as, e.g., a ZFP/FokI fusion)require the use of two such fusion molecules, each generally directed toa distinct target sequence. Target sequences for the two fusion proteinscan be chosen so that targeted cleavage is directed to a unique site ina genome, as discussed above. A potential source of reduced cleavagespecificity could result from homodimerization of one of the twoZFP/cleavage half-domain fusions. This might occur, for example, due tothe presence, in a genome, of inverted repeats of the target sequencesfor one of the two ZFP/cleavage half-domain fusions, located so as toallow two copies of the same fusion protein to bind with an orientationand spacing that allows formation of a functional dimer.

One approach for reducing the probability of this type of aberrantcleavage at sequences other than the intended target site involvesgenerating variants of the cleavage half-domain that minimize or preventhomodimerization. Preferably, one or more amino acids in the region ofthe half-domain involved in its dimerization are altered. In the crystalstructure of the FokI protein dimer, the structure of the cleavagehalf-domains is reported to be similar to the arrangement of thecleavage half-domains during cleavage of DNA by FokI. Wah et al. (1998)Proc. Natl. Acad. Sci. USA 95:10564-10569. This structure indicates thatamino acid residues at positions 483 and 487 play a key role in thedimerization of the FokI cleavage half-domains. The structure alsoindicates that amino acid residues at positions 446, 447, 479, 483, 484,486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 are allclose enough to the dimerization interface to influence dimerization.Accordingly, amino acid sequence alterations at one or more of theaforementioned positions will likely alter the dimerization propertiesof the cleavage half-domain. Such changes can be introduced, forexample, by constructing a library containing (or encoding) differentamino acid residues at these positions and selecting variants with thedesired properties, or by rationally designing individual mutants. Inaddition to preventing homodimerization, it is also possible that someof these mutations may increase the cleavage efficiency above thatobtained with two wild-type cleavage half-domains.

Accordingly, alteration of a FokI cleavage half-domain at any amino acidresidue which affects dimerization can be used to prevent one of a pairof ZFP/FokI fusions from undergoing homodimerization which can lead tocleavage at undesired sequences. Thus, for targeted cleavage using apair of ZFP/FokI fusions, one or both of the fusion proteins cancomprise one or more amino acid alterations that inhibitself-dimerization, but allow heterodimerization of the two fusionproteins to occur such that cleavage occurs at the desired target site.In certain embodiments, alterations are present in both fusion proteins,and the alterations have additive effects; i.e., homodimerization ofeither fusion, leading to aberrant cleavage, is minimized or abolished,while heterodimerization of the two fusion proteins is facilitatedcompared to that obtained with wild-type cleavage half-domains. SeeExample 5.

Methods for Targeted Alteration of Genomic Sequences and TargetedRecombination

Also described herein are methods of replacing a genomic sequence (e.g.,a region of interest in cellular chromatin) with a homologousnon-identical sequence (i.e., targeted recombination). Previous attemptsto replace particular sequences have involved contacting a cell with apolynucleotide comprising sequences bearing homology to a chromosomalregion (i.e., a donor DNA), followed by selection of cells in which thedonor DNA molecule had undergone homologous recombination into thegenome. The success rate of these methods is low, due to poor efficiencyof homologous recombination and a high frequency of non-specificinsertion of the donor DNA into regions of the genome other than thetarget site.

The present disclosure provides methods of targeted sequence alterationcharacterized by a greater efficiency of targeted recombination and alower frequency of non-specific insertion events. The methods involvemaking and using engineered zinc finger binding domains fused tocleavage domains (or cleavage half-domains) to make one or more targeteddouble-stranded breaks in cellular DNA. Because double-stranded breaksin cellular DNA stimulate homologous recombination several thousand-foldin the vicinity of the cleavage site, such targeted cleavage allows forthe alteration or replacement (via homologous recombination) ofsequences at virtually any site in the genome.

In addition to the fusion molecules described herein, targetedreplacement of a selected genomic sequence also requires theintroduction of the replacement (or donor) sequence. The donor sequencecan be introduced into the cell prior to, concurrently with, orsubsequent to, expression of the fusion protein(s). The donorpolynucleotide contains sufficient homology to a genomic sequence tosupport homologous recombination between it and the genomic sequence towhich it bears homology. Approximately 25, 50 100 or 200 nucleotides ormore of sequence homology between a donor and a genomic sequence (or anyintegral value between 10 and 200 nucleotides, or more) will supporthomologous recombination therebetween. Donor sequences can range inlength from 10 to 5,000 nucleotides (or any integral value ofnucleotides therebetween) or longer. It will be readily apparent thatthe donor sequence is typically not identical to the genomic sequencethat it replaces. For example, the sequence of the donor polynucleotidecan contain one or more single base changes, insertions, deletions,inversions or rearrangements with respect to the genomic sequence, solong as sufficient homology is present to support homologousrecombination. Alternatively, a donor sequence can contain anon-homologous sequence flanked by two regions of homology.Additionally, donor sequences can comprise a vector molecule containingsequences that are not homologous to the region of interest in cellularchromatin. Generally, the homologous region(s) of a donor sequence willhave at least 50% sequence identity to a genomic sequence with whichrecombination is desired. In certain embodiments, 60%, 70%, 80%, 90%,95%, 98%, 99%, or 99.9% sequence identity is present. Any value between1% and 100% sequence identity can be present, depending upon the lengthof the donor polynucleotide.

A donor molecule can contain several, discontinuous regions of homologyto cellular chromatin. For example, for targeted insertion of sequencesnot normally present in a region of interest, said sequences can bepresent in a donor nucleic acid molecule and flanked by regions ofhomology to sequence in the region of interest.

To simplify assays (e.g., hybridization, PCR, restriction enzymedigestion) for determining successful insertion of the donor sequence,certain sequence differences may be present in the donor sequence ascompared to the genomic sequence. Preferably, if located in a codingregion, such nucleotide sequence differences will not change the aminoacid sequence, or will make silent amino acid changes (i.e., changeswhich do not affect the structure or function of the protein). The donorpolynucleotide can optionally contain changes in sequences correspondingto the zinc finger domain binding sites in the region of interest, toprevent cleavage of donor sequences that have been introduced intocellular chromatin by homologous recombination.

The donor polynucleotide can be DNA or RNA, single-stranded ordouble-stranded and can be introduced into a cell in linear or circularform. If introduced in linear form, the ends of the donor sequence canbe protected (e.g., from exonucleolytic degradation) by methods known tothose of skill in the art. For example, one or more dideoxynucleotideresidues are added to the 3′ terminus of a linear molecule and/orself-complementary oligonucleotides are ligated to one or both ends.See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additionalmethods for protecting exogenous polynucleotides from degradationinclude, but are not limited to, addition of terminal amino group(s) andthe use of modified internucleotide linkages such as, for example,phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyriboseresidues. A polynucleotide can be introduced into a cell as part of avector molecule having additional sequences such as, for example,replication origins, promoters and genes encoding antibiotic resistance.Moreover, donor polynucleotides can be introduced as naked nucleic acid,as nucleic acid complexed with an agent such as a liposome or poloxamer,or can be delivered by viruses (e.g., adenovirus, AAV).

Without being bound by one theory, it appears that the presence of adouble-stranded break in a cellular sequence, coupled with the presenceof an exogenous DNA molecule having homology to a region adjacent to orsurrounding the break, activates cellular mechanisms which repair thebreak by transfer of sequence information from the donor molecule intothe cellular (e.g., genomic or chromosomal) sequence; i.e., by aprocesses of homologous recombination. Applicants' methodsadvantageously combine the powerful targeting capabilities of engineeredZFPs with a cleavage domain (or cleavage half-domain) to specificallytarget a double-stranded break to the region of the genome at whichrecombination is desired.

For alteration of a chromosomal sequence, it is not necessary for theentire sequence of the donor to be copied into the chromosome, as longas enough of the donor sequence is copied to effect the desired sequencealteration.

The efficiency of insertion of donor sequences by homologousrecombination is inversely related to the distance, in the cellular DNA,between the double-stranded break and the site at which recombination isdesired. In other words, higher homologous recombination efficienciesare observed when the double-stranded break is closer to the site atwhich recombination is desired. In cases in which a precise site ofrecombination is not predetermined (e.g., the desired recombinationevent can occur over an interval of genomic sequence), the length andsequence of the donor nucleic acid, together with the site(s) ofcleavage, are selected to obtain the desired recombination event. Incases in which the desired event is designed to change the sequence of asingle nucleotide pair in a genomic sequence, cellular chromatin iscleaved within 10,000 nucleotides on either side of that nucleotidepair. In certain embodiments, cleavage occurs within 500, 200, 100, 90,80, 70, 60, 50, 40, 30, 20, 10, 5, or 2 nucleotides, or any integralvalue between 2 and 1,000 nucleotides, on either side of the nucleotidepair whose sequence is to be changed.

As detailed above, the binding sites for two fusion proteins, eachcomprising a zinc finger binding domain and a cleavage half-domain, canbe located 5-8 or 15-18 nucleotides apart, as measured from the edge ofeach binding site nearest the other binding site, and cleavage occursbetween the binding sites. Whether cleavage occurs at a single site orat multiple sites between the binding sites is immaterial, since thecleaved genomic sequences are replaced by the donor sequences. Thus, forefficient alteration of the sequence of a single nucleotide pair bytargeted recombination, the midpoint of the region between the bindingsites is within 10,000 nucleotides of that nucleotide pair, preferablywithin 1,000 nucleotides, or 500 nucleotides, or 200 nucleotides, or 100nucleotides, or 50 nucleotides, or 20 nucleotides, or 10 nucleotides, or5 nucleotide, or 2 nucleotides, or one nucleotide, or at the nucleotidepair of interest.

In certain embodiments, a homologous chromosome can serve as the donorpolynucleotide. Thus, for example, correction of a mutation in aheterozygote can be achieved by engineering fusion proteins which bindto and cleave the mutant sequence on one chromosome, but do not cleavethe wild-type sequence on the homologous chromosome. The double-strandedbreak on the mutation-bearing chromosome stimulates a homology-based“gene conversion” process in which the wild-type sequence from thehomologous chromosome is copied into the cleaved chromosome, thusrestoring two copies of the wild-type sequence.

Methods and compositions are also provided that may enhance levels oftargeted recombination including, but not limited to, the use ofadditional ZFP-functional domain fusions to activate expression of genesinvolved in homologous recombination, such as, for example, members ofthe RAD52 epistasis group (e.g., Rad50, Rad51, Rad51B, Rad51C, Rad51D,Rad52, Rad54, Rad54B, Mre11, XRCC2, XRCC3), genes whose productsinteract with the aforementioned gene products (e.g., BRCA1, BRCA2)and/or genes in the NBS1 complex. Similarly ZFP-functional domainfusions can be used, in combination with the methods and compositionsdisclosed herein, to repress expression of genes involved innon-homologous end joining (e.g., Ku70/80, XRCC4, poly(ADP ribose)polymerase, DNA ligase 4). See, for example, Yanez et al. (1998) GeneTherapy 5:149-159; Hoeijmakers (2001) Nature 411:366-374; Johnson et al.(2001) Biochem. Soc. Trans. 29:196-201; Tauchi et al. (2002) Oncogene21:8967-8980. Methods for activation and repression of gene expressionusing fusions between a zinc finger binding domain and a functionaldomain are disclosed in co-owned U.S. Pat. No. 6,534,261. Additionalrepression methods include the use of antisense oligonucleotides and/orsmall interfering RNA (siRNA or RNAi) targeted to the sequence of thegene to be repressed.

As an alternative to or, in addition to, activating expression of geneproducts involved in homologous recombination, fusions of these protein(or functional fragments thereof) with a zinc finger binding domaintargeted to the region of interest, can be used to recruit theseproteins (recombination proteins) to the region of interest, therebyincreasing their local concentration and further stimulating homologousrecombination processes. Alternatively, a polypeptide involved inhomologous recombination as described above (or a functional fragmentthereof) can be part of a triple fusion protein comprising a zinc fingerbinding domain, a cleavage domain (or cleavage half-domain) and therecombination protein (or functional fragment thereof). Additionalproteins involved in gene conversion and recombination-related chromatinremodeling, which can be used in the aforementioned methods andcompositions, include histone acetyltransferases (e.g., Esa1p, Tip60),histone methyltransferases (e.g., Dot1p), histone kinases and histonephosphatases.

The p53 protein has been reported to play a central role in repressinghomologous recombination (HR). See, for example, Valerie et al., (2003)Oncogene 22:5792-5812; Janz, et al. (2002) Oncogene 21:5929-5933. Forexample, the rate of HR in p53-deficient human tumor lines is10,000-fold greater than in primary human fibroblasts, and there is a100-fold increase in HR in tumor cells with a non-functional p53compared to those with functional p53. Mekeel et al. (1997) Oncogene14:1847-1857. In addition, overexpression of p53 dominant negativemutants leads to a 20-fold increase in spontaneous recombination.Bertrand et al. (1997) Oncogene 14:1117-1122. Analysis of different p53mutations has revealed that the roles of p53 in transcriptionaltransactivation and G1 cell cycle checkpoint control are separable fromits involvement in H R. Saintigny et al. (1999) Oncogene 18:3553-3563;Boehden et al. (2003) Oncogene 22:4111-4117. Accordingly, downregulationof p53 activity can serve to increase the efficiency of targetedhomologous recombination using the methods and compositions disclosedherein. Any method for downregulation of p53 activity can be used,including but not limited to cotransfection and overexpression of a p53dominant negative mutant or targeted repression of p53 gene expressionaccording to methods disclosed, e.g., in co-owned U.S. Pat. No.6,534,261.

Further increases in efficiency of targeted recombination, in cellscomprising a zinc finger/nuclease fusion molecule and a donor DNAmolecule, are achieved by blocking the cells in the G₂ phase of the cellcycle, when homology-driven repair processes are maximally active. Sucharrest can be achieved in a number of ways. For example, cells can betreated with e.g., drugs, compounds and/or small molecules whichinfluence cell-cycle progression so as to arrest cells in G₂ phase.Exemplary molecules of this type include, but are not limited to,compounds which affect microtubule polymerization (e.g., vinblastine,nocodazole, Taxol), compounds that interact with DNA (e.g.,cis-platinum(II) diamine dichloride, Cisplatin, doxorubicin) and/orcompounds that affect DNA synthesis (e.g., thymidine, hydroxyurea,L-mimosine, etoposide, 5-fluorouracil). Additional increases inrecombination efficiency are achieved by the use of histone deacetylase(HDAC) inhibitors (e.g., sodium butyrate, trichostatin A) which alterchromatin structure to make genomic DNA more accessible to the cellularrecombination machinery.

Additional methods for cell-cycle arrest include overexpression ofproteins which inhibit the activity of the CDK cell-cycle kinases, forexample, by introducing a cDNA encoding the protein into the cell or byintroducing into the cell an engineered ZFP which activates expressionof the gene encoding the protein. Cell-cycle arrest is also achieved byinhibiting the activity of cyclins and CDKs, for example, using RNAimethods (e.g., U.S. Pat. No. 6,506,559) or by introducing into the cellan engineered ZFP which represses expression of one or more genesinvolved in cell-cycle progression such as, for example, cyclin and/orCDK genes. See, e.g., co-owned U.S. Pat. No. 6,534,261 for methods forthe synthesis of engineered zinc finger proteins for regulation of geneexpression.

Alternatively, in certain cases, targeted cleavage is conducted in theabsence of a donor polynucleotide (preferably in S or G₂ phase), andrecombination occurs between homologous chromosomes.

Methods to Screen for Cellular Factors that Facilitate HomologousRecombination

Since homologous recombination is a multi-step process requiring themodification of DNA ends and the recruitment of several cellular factorsinto a protein complex, the addition of one or more exogenous factors,along with donor DNA and vectors encoding zinc finger-cleavage domainfusions, can be used to facilitate targeted homologous recombination. Anexemplary method for identifying such a factor or factors employsanalyses of gene expression using microarrays (e.g., Affymetrix GeneChip® arrays) to compare the mRNA expression patterns of differentcells. For example, cells that exhibit a higher capacity, to stimulatedouble strand break-driven homologous recombination in the presence ofdonor DNA and zinc finger-cleavage domain fusions, either unaided orunder conditions known to increase the level of gene correction, can beanalyzed for their gene expression patterns compared to cells that lacksuch capacity. Genes that are upregulated or downregulated in a mannerthat directly correlates with increased levels of homologousrecombination are thereby identified and can be cloned into any one of anumber of expression vectors. These expression constructs can beco-transfected along with zinc finger-cleavage domain fusions and donorconstructs to yield improved methods for achieving high-efficiencyhomologous recombination. Alternatively, expression of such genes can beappropriately regulated using engineered zinc finger roteins whichmodulate expression (either activation or repression) of one or morethese genes. See, e.g., co-owned U.S. Pat. No. 6,534,261 for methods forthe synthesis of engineered zinc finger proteins for regulation of geneexpression.

As an example, it was observed that the different clones obtained in theexperiments described in Example 9 and FIG. 27 exhibited a wide-range ofhomologous recombination frequencies, when transfected with donor DNAand plasmids encoding zinc finger-cleavage domain fusions. Geneexpression in clones showing a high frequency of targeted recombinationcan thus be compared to that in clones exhibiting a low frequency, andexpression patterns unique to the former clones can be identified.

As an additional example, studies using cell cycle inhibitors (e.g.,nocodazole or vinblastine, see e.g., Examples 11, 14 and 15) showed thatcells arrested in the G2 phase of the cell cycle carried out homologousrecombination at higher rates, indicating that cellular factorsresponsible for homologous recombination may be preferentially expressedor active in G2. One way to identify these factors is to compare themRNA expression patterns between the stably transfected HEK 293 cellclones that carry out gene correction at high and low levels (e.g.,clone T18 vs. clone T7). Similar comparisons are made between these celllines in response to compounds that arrest the cells in G2 phase.Candidate genes that are differentially expressed in cells that carryout homologous recombination at a higher rate, either unaided or inresponse to compounds that arrest the cells in G2, are identified,cloned, and re-introduced into cells to determine whether theirexpression is sufficient to re-capitulate the improved rates.Alternatively, expression of said candidate genes is activated usingengineered zinc finger transcription factors as described, for example,in co-owned U.S. Pat. No. 6,534,261.

Expression Vectors

A nucleic acid encoding one or more ZFPs or ZFP fusion proteins can becloned into a vector for transformation into prokaryotic or eukaryoticcells for replication and/or expression. Vectors can be prokaryoticvectors, e.g., plasmids, or shuttle vectors, insect vectors, oreukaryotic vectors. A nucleic acid encoding a ZFP can also be clonedinto an expression vector, for administration to a plant cell, animalcell, preferably a mammalian cell or a human cell, fungal cell,bacterial cell, or protozoal cell.

To obtain expression of a cloned gene or nucleic acid, sequencesencoding a ZFP or ZFP fusion protein are typically subcloned into anexpression vector that contains a promoter to direct transcription.Suitable bacterial and eukaryotic promoters are well known in the artand described, e.g., in Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989; 3^(rd) ed., 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., supra. Bacterial expression systemsfor expressing the ZFP are available in, e.g., E. coli, Bacillus sp.,and Salmonella (Palva et al., Gene 22:229-235 (1983)). Kits for suchexpression systems are commercially available. Eukaryotic expressionsystems for mammalian cells, yeast, and insect cells are well known bythose of skill in the art and are also commercially available.

The promoter used to direct expression of a ZFP-encoding nucleic aciddepends on the particular application. For example, a strongconstitutive promoter is typically used for expression and purificationof ZFP. In contrast, when a ZFP is administered in vivo for generegulation, either a constitutive or an inducible promoter is used,depending on the particular use of the ZFP. In addition, a preferredpromoter for administration of a ZFP can be a weak promoter, such as HSVTK or a promoter having similar activity. The promoter typically canalso include elements that are responsive to transactivation, e.g.,hypoxia response elements, Gal4 response elements, lac repressorresponse element, and small molecule control systems such astet-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard,PNAS 89:5547 (1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wanget al., Gene Ther. 4:432-441 (1997); Neering et al., Blood 88:1147-1155(1996); and Rendahl et al., Nat. Biotechnol. 16:757-761 (1998)). TheMNDU3 promoter can also be used, and is preferentially active in CD34⁺hematopoietic stem cells.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the nucleic acid inhost cells, either prokaryotic or eukaryotic. A typical expressioncassette thus contains a promoter operably linked, e.g., to a nucleicacid sequence encoding the ZFP, and signals required, e.g., forefficient polyadenylation of the transcript, transcriptionaltermination, ribosome binding sites, or translation termination.Additional elements of the cassette may include, e.g., enhancers, andheterologous splicing signals.

The particular expression vector used to transport the geneticinformation into the cell is selected with regard to the intended use ofthe ZFP, e.g., expression in plants, animals, bacteria, fungus,protozoa, etc. (see expression vectors described below). Standardbacterial expression vectors include plasmids such as pBR322-basedplasmids, pSKF, pET23D, and commercially available fusion expressionsystems such as GST and LacZ. An exemplary fusion protein is the maltosebinding protein, “MBP.” Such fusion proteins are used for purificationof the ZFP. Epitope tags can also be added to recombinant proteins toprovide convenient methods of isolation, for monitoring expression, andfor monitoring cellular and subcellular localization, e.g., c-myc orFLAG.

Expression vectors containing regulatory elements from eukaryoticviruses are often used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+,pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 late promoter, metallothionein promoter, murine mammary tumor viruspromoter, Rous sarcoma virus promoter, polyhedrin promoter, or otherpromoters shown effective for expression in eukaryotic cells.

Some expression systems have markers for selection of stably transfectedcell lines such as thymidine kinase, hygromycin B phosphotransferase,and dihydrofolate reductase. High yield expression systems are alsosuitable, such as using a baculovirus vector in insect cells, with a ZFPencoding sequence under the direction of the polyhedrin promoter orother strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of recombinant sequences.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of protein,which are then purified using standard techniques (see, e.g., Colley etal., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,ultrasonic methods (e.g., sonoporation), liposomes, microinjection,naked DNA, plasmid vectors, viral vectors, both episomal andintegrative, and any of the other well known methods for introducingcloned genomic DNA, cDNA, synthetic DNA or other foreign geneticmaterial into a host cell (see, e.g., Sambrook et al., supra). It isonly necessary that the particular genetic engineering procedure used becapable of successfully introducing at least one gene into the host cellcapable of expressing the protein of choice.

Nucleic Acids Encoding Fusion Proteins and Delivery to Cells

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered ZFPs in cells (e.g.,mammalian cells) and target tissues. Such methods can also be used toadminister nucleic acids encoding ZFPs to cells in vitro. In certainembodiments, nucleic acids encoding ZFPs are administered for in vivo orex vivo gene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome or poloxamer. Viral vector delivery systemsinclude DNA and RNA viruses, which have either episomal or integratedgenomes after delivery to the cell. For a review of gene therapyprocedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner,TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology Doerfler and Böhm (eds) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids encoding engineered ZFPsinclude electroporation, lipofection, microinjection, biolistics,virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acidconjugates, naked DNA, artificial virions, and agent-enhanced uptake ofDNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) canalso be used for delivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.) and BTX Molecular Delivery Systems (Holliston, Mass.).

Lipofection is described in e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No.4,946,787; and U.S. Pat. No. 4,897,355) and lipofection reagents aresold commercially (e.g., Transfectam™ and Lipofectin™). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Felgner, WO 91/17424, WO91/16024. Delivery can be to cells (ex vivo administration) or targettissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs take advantage of highly evolvedprocesses for targeting a virus to specific cells in the body andtrafficking the viral payload to the nucleus. Viral vectors can beadministered directly to patients (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to patients (exvivo). Conventional viral based systems for the delivery of ZFPsinclude, but are not limited to, retroviral, lentivirus, adenoviral,adeno-associated, vaccinia and herpes simplex virus vectors for genetransfer. Integration in the host genome is possible with theretrovirus, lentivirus, and adeno-associated virus gene transfermethods, often resulting in long term expression of the insertedtransgene. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SW), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression of a ZFP fusion protein ispreferred, adenoviral based systems can be used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand high levels of expression have been obtained. This vector can beproduced in large quantities in a relatively simple system.Adeno-associated virus (“AAV”) vectors are also used to transduce cellswith target nucleic acids, e.g., in the in vitro production of nucleicacids and peptides, and for in vivo and ex vivo gene therapy procedures(see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No.4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994);Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinantAAV vectors are described in a number of publications, including U.S.Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260(1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat& Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette.

Efficient gene transfer and stable transgene delivery due to integrationinto the genomes of the transduced cell are key features for this vectorsystem. (Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al.,Gene Ther. 9:748-55 (1996)).

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a ZFPnucleic acid (gene or cDNA), and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med.176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1(granulocytes), and Iad (differentiated antigen presenting cells) (seeInaba et al., J. Exp. Med. 176:1693-1702 (1992)).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic ZFP nucleic acids can also be administered directly to anorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Methods for introduction of DNA into hematopoietic stem cells aredisclosed, for example, in U.S. Pat. No. 5,928,638.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

DNA constructs may be introduced into the genome of a desired plant hostby a variety of conventional techniques. For reviews of such techniquessee, for example, Weissbach & Weissbach Methods for Plant. MolecularBiology (1988, Academic Press, N.Y.) Section VIII, pp. 421-463; andGrierson & Corey, Plant Molecular Biology (1988, 2d Ed.), Blackie,London, Ch. 7-9. For example, the DNA construct may be introduceddirectly into the genomic DNA of the plant cell using techniques such aselectroporation and microinjection of plant cell protoplasts, or the DNAconstructs can be introduced directly to plant tissue using biolisticmethods, such as DNA particle bombardment (see, e.g., Klein et al (1987)Nature 327:70-73). Alternatively, the DNA constructs may be combinedwith suitable T-DNA flanking regions and introduced into a conventionalAgrobacterium tumefaciens host vector. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, for example Horsch et al (1984) Science 233:496-498, and Fraley etal (1983) Proc. Nat'l. Acad. Sci. USA 80:4803. The virulence functionsof the Agrobacterium tumefaciens host will direct the insertion of theconstruct and adjacent marker into the plant cell DNA when the cell isinfected by the bacteria using binary T DNA vector (Bevan (1984) Nuc.Acid Res. 12:8711-8721) or the co-cultivation procedure (Horsch et al(1985) Science 227:1229-1231). Generally, the Agrobacteriumtransformation system is used to engineer dicotyledonous plants (Bevanet al (1982) Ann. Rev. Genet. 16:357-384; Rogers et al (1986) MethodsEnzymol. 118:627-641). The Agrobacterium transformation system may alsobe used to transform, as well as transfer, DNA to monocotyledonousplants and plant cells. See Hernalsteen et al (1984) EMBO J.3:3039-3041; Hooykass-Van Slogteren et al (1984) Nature 311:763-764;Grimsley et al (1987) Nature 325:1677-179; Boulton et al (1989) PlantMol. Biol. 12:31-40; and Gould et al (1991) Plant Physiol. 95:426-434.

Alternative gene transfer and transformation methods include, but arenot limited to, protoplast transformation through calcium-, polyethyleneglycol (PEG)- or electroporation-mediated uptake of naked DNA (seePaszkowski et al. (1984) EMBO J. 3:2717-2722, Potrykus et al. (1985)Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc. Nat. Acad.Sci. USA 82:5824-5828; and Shimamoto (1989) Nature 338:274-276) andelectroporation of plant tissues (D'Halluin et al. (1992) Plant Cell4:1495-1505). Additional methods for plant cell transformation includemicroinjection, silicon carbide mediated DNA uptake (Kaeppler et al.(1990) Plant Cell Reporter 9:415-418), and microprojectile bombardment(see Klein et al. (1988) Proc. Nat. Acad. Sci. USA 85:4305-4309; andGordon-Kamm et al. (1990) Plant Cell 2:603-618).

The disclosed methods and compositions can be used to insert exogenoussequences into a predetermined location in a plant cell genome. This isuseful inasmuch as expression of an introduced transgene into a plantgenome depends critically on its integration site. Accordingly, genesencoding, e.g., nutrients, antibiotics or therapeutic molecules can beinserted, by targeted recombination, into regions of a plant genomefavorable to their expression.

Transformed plant cells which are produced by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker which has been introduced together withthe desired nucleotide sequences. Plant regeneration from culturedprotoplasts is described in Evans, et al., “Protoplasts Isolation andCulture” in Handbook of Plant Cell Culture, pp. 124-176, MacmillianPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, pollens,embryos or parts thereof. Such regeneration techniques are describedgenerally in Klee et al (1987) Ann. Rev. of Plant Phys. 38:467-486.

Nucleic acids introduced into a plant cell can be used to confer desiredtraits on essentially any plant. A wide variety of plants and plant cellsystems may be engineered for the desired physiological and agronomiccharacteristics described herein using the nucleic acid constructs ofthe present disclosure and the various transformation methods mentionedabove. In preferred embodiments, target plants and plant cells forengineering include, but are not limited to, those monocotyledonous anddicotyledonous plants, such as crops including grain crops (e.g., wheat,maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear,strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops(e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g.,lettuce, spinach); flowering plants (e.g., petunia, rose,chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plantsused in phytoremediation (e.g., heavy metal accumulating plants); oilcrops (e.g., sunflower, rape seed) and plants used for experimentalpurposes (e.g., Arabidopsis). Thus, the disclosed methods andcompositions have use over a broad range of plants, including, but notlimited to, species from the genera Asparagus, Avena, Brassica, Citrus,Citrullus, Capsicum, Cucurbita, Daucus, Glycine, Hordeum, Lactuca,Lycopersicon, Malus, Manihot, Nicotiana, Oryza, Persea, Pisum, Pyrus,Prunus, Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna, andZea.

One of skill in the art will recognize that after the expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

A transformed plant cell, callus, tissue or plant may be identified andisolated by selecting or screening the engineered plant material fortraits encoded by the marker genes present on the transforming DNA. Forinstance, selection may be performed by growing the engineered plantmaterial on media containing an inhibitory amount of the antibiotic orherbicide to which the transforming gene construct confers resistance.Further, transformed plants and plant cells may also be identified byscreening for the activities of any visible marker genes (e.g., theβ-glucuronidase, luciferase, B or C1 genes) that may be present on therecombinant nucleic acid constructs. Such selection and screeningmethodologies are well known to those skilled in the art.

Physical and biochemical methods also may be used to identify plant orplant cell transformants containing inserted gene constructs. Thesemethods include but are not limited to: 1) Southern analysis or PCRamplification for detecting and determining the structure of therecombinant DNA insert; 2) Northern blot, S1 RNase protection,primer-extension or reverse transcriptase-PCR amplification fordetecting and examining RNA transcripts of the gene constructs; 3)enzymatic assays for detecting enzyme or ribozyme activity, where suchgene products are encoded by the gene construct; 4) protein gelelectrophoresis, Western blot techniques, immunoprecipitation, orenzyme-linked immunoassays, where the gene construct products areproteins. Additional techniques, such as in situ hybridization, enzymestaining, and immunostaining, also may be used to detect the presence orexpression of the recombinant construct in specific plant organs andtissues. The methods for doing all these assays are well known to thoseskilled in the art.

Effects of gene manipulation using the methods disclosed herein can beobserved by, for example, northern blots of the RNA (e.g., mRNA)isolated from the tissues of interest. Typically, if the amount of mRNAhas increased, it can be assumed that the corresponding endogenous geneis being expressed at a greater rate than before. Other methods ofmeasuring gene and/or CYP74B activity can be used. Different types ofenzymatic assays can be used, depending on the substrate used and themethod of detecting the increase or decrease of a reaction product orby-product. In addition, the levels of and/or CYP74B protein expressedcan be measured immunochemically, i.e., ELISA, RIA, EIA and otherantibody based assays well known to those of skill in the art, such asby electrophoretic detection assays (either with staining or westernblotting). The transgene may be selectively expressed in some tissues ofthe plant or at some developmental stages, or the transgene may beexpressed in substantially all plant tissues, substantially along itsentire life cycle. However, any combinatorial expression mode is alsoapplicable.

The present disclosure also encompasses seeds of the transgenic plantsdescribed above wherein the seed has the transgene or gene construct.The present disclosure further encompasses the progeny, clones, celllines or cells of the transgenic plants described above wherein saidprogeny, clone, cell line or cell has the transgene or gene construct.

Delivery Vehicles

An important factor in the administration of polypeptide compounds, suchas ZFP fusion proteins, is ensuring that the polypeptide has the abilityto traverse the plasma membrane of a cell, or the membrane of anintra-cellular compartment such as the nucleus. Cellular membranes arecomposed of lipid-protein bilayers that are freely permeable to small,nonionic lipophilic compounds and are inherently impermeable to polarcompounds, macromolecules, and therapeutic or diagnostic agents.However, proteins and other compounds such as liposomes have beendescribed, which have the ability to translocate polypeptides such asZFPs across a cell membrane.

For example, “membrane translocation polypeptides” have amphiphilic orhydrophobic amino acid subsequences that have the ability to act asmembrane-translocating carriers. In one embodiment, homeodomain proteinshave the ability to translocate across cell membranes. The shortestinternalizable peptide of a homeodomain protein, Antennapedia, was foundto be the third helix of the protein, from amino acid position 43 to 58(see, e.g., Prochiantz, Current Opinion in Neurobiology 6:629-634(1996)). Another subsequence, the h (hydrophobic) domain of signalpeptides, was found to have similar cell membrane translocationcharacteristics (see, e.g., Lin et al., J. Biol. Chem. 270:1 4255-14258(1995)).

Examples of peptide sequences which can be linked to a protein, forfacilitating uptake of the protein into cells, include, but are notlimited to: an 11 amino acid peptide of the tat protein of HIV; a 20residue peptide sequence which corresponds to amino acids 84-103 of thep16 protein (see Fahraeus et al., Current Biology 6:84 (1996)); thethird helix of the 60-amino acid long homeodomain of Antennapedia(Derossi et al., J. Biol. Chem. 269:10444 (1994)); the h region of asignal peptide such as the Kaposi fibroblast growth factor (K-FGF) hregion (Lin et al., supra); or the VP22 translocation domain from HSV(Elliot & O'Hare, Cell 88:223-233 (1997)). Other suitable chemicalmoieties that provide enhanced cellular uptake may also be chemicallylinked to ZFPs. Membrane translocation domains (i.e., internalizationdomains) can also be selected from libraries of randomized peptidesequences. See, for example, Yeh et al. (2003) Molecular Therapy7(5):S461, Abstract #1191.

Toxin molecules also have the ability to transport polypeptides acrosscell membranes. Often, such molecules (called “binary toxins”) arecomposed of at least two parts: a translocation/binding domain orpolypeptide and a separate toxin domain or polypeptide. Typically, thetranslocation domain or polypeptide binds to a cellular receptor, andthen the toxin is transported into the cell. Several bacterial toxins,including Clostridium perfringens iota toxin, diphtheria toxin (DT),Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus anthracistoxin, and pertussis adenylate cyclase (CYA), have been used to deliverpeptides to the cell cytosol as internal or amino-terminal fusions(Arora et al., J. Biol. Chem., 268:3334-3341 (1993); Perelle et al.,Infect. Immun., 61:5147-5156 (1993); Stenmark et al., J. Cell Biol.113:1025-1032 (1991); Donnelly et al., PNAS 90:3530-3534 (1993);Carbonetti et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 95:295 (1995);Sebo et al., Infect. Immun. 63:3851-3857 (1995); Klimpel et al., PNASU.S.A. 89:10277-10281 (1992); and Novak et al., J. Biol. Chem.267:17186-17193 1992)).

Such peptide sequences can be used to translocate ZFPs across a cellmembrane. ZFPs can be conveniently fused to or derivatized with suchsequences. Typically, the translocation sequence is provided as part ofa fusion protein. Optionally, a linker can be used to link the ZFP andthe translocation sequence. Any suitable linker can be used, e.g., apeptide linker.

The ZFP can also be introduced into an animal cell, preferably amammalian cell, via a liposomes and liposome derivatives such asimmunoliposomes. The term “liposome” refers to vesicles comprised of oneor more concentrically ordered lipid bilayers, which encapsulate anaqueous phase. The aqueous phase typically contains the compound to bedelivered to the cell, i.e., a ZFP.

The liposome fuses with the plasma membrane, thereby releasing the druginto the cytosol. Alternatively, the liposome is phagocytosed or takenup by the cell in a transport vesicle. Once in the endosome orphagosome, the liposome either degrades or fuses with the membrane ofthe transport vesicle and releases its contents.

In current methods of drug delivery via liposomes, the liposomeultimately becomes permeable and releases the encapsulated compound (inthis case, a ZFP) at the target tissue or cell. For systemic or tissuespecific delivery, this can be accomplished, for example, in a passivemanner wherein the liposome bilayer degrades over time through theaction of various agents in the body. Alternatively, active drug releaseinvolves using an agent to induce a permeability change in the liposomevesicle. Liposome membranes can be constructed so that they becomedestabilized when the environment becomes acidic near the liposomemembrane (see, e.g., PNAS 84:7851 (1987); Biochemistry 28:908 (1989)).When liposomes are endocytosed by a target cell, for example, theybecome destabilized and release their contents. This destabilization istermed fusogenesis. Dioleoylphosphatidylethanolamine (DOPE) is the basisof many “fusogenic” systems.

Such liposomes typically comprise a ZFP and a lipid component, e.g., aneutral and/or cationic lipid, optionally including areceptor-recognition molecule such as an antibody that binds to apredetermined cell surface receptor or ligand (e.g., an antigen). Avariety of methods are available for preparing liposomes as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054,4,501,728, 4,774,085, 4,837,028, 4,235,871, 4,261,975, 4,485,054,4,501,728, 4,774,085, 4,837,028, 4,946,787, PCT Publication No. WO91\17424, Deamer & Bangham, Biochim. Biophys. Acta 443:629-634 (1976);Fraley, et al., PNAS 76:3348-3352 (1979); Hope et al., Biochim. Biophys.Acta 812:55-65 (1985); Mayer et al., Biochim. Biophys. Acta 858:161-168(1986); Williams et al., PNAS 85:242-246 (1988); Liposomes (Ostro (ed.),1983, Chapter 1); Hope et al., Chem. Phys. Lip. 40:89 (1986);Gregoriadis, Liposome Technology (1984) and Lasic, Liposomes: fromPhysics to Applications (1993)). Suitable methods include, for example,sonication, extrusion, high pressure/homogenization, microfluidization,detergent dialysis, calcium-induced fusion of small liposome vesiclesand ether-fusion methods, all of which are known to those of skill inthe art.

In certain embodiments, it is desirable to target liposomes usingtargeting moieties that are specific to a particular cell type, tissue,and the like. Targeting of liposomes using a variety of targetingmoieties (e.g., ligands, receptors, and monoclonal antibodies) has beendescribed. See, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044.

Examples of targeting moieties include monoclonal antibodies specific toantigens associated with neoplasms, such as prostate cancer specificantigen and MAGE. Tumors can also be diagnosed by detecting geneproducts resulting from the activation or over-expression of oncogenes,such as ras or c-erbB2. In addition, many tumors express antigensnormally expressed by fetal tissue, such as the alphafetoprotein (AFP)and carcinoembryonic antigen (CEA). Sites of viral infection can bediagnosed using various viral antigens such as hepatitis B core andsurface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Barr virusantigens, human immunodeficiency type-1 virus (HIV1) and papilloma virusantigens. Inflammation can be detected using molecules specificallyrecognized by surface molecules which are expressed at sites ofinflammation such as integrins (e.g., VCAM-1), selectin receptors (e.g.,ELAM-1) and the like.

Standard methods for coupling targeting agents to liposomes can be used.These methods generally involve incorporation into liposomes of lipidcomponents, e.g., phosphatidylethanolamine, which can be activated forattachment of targeting agents, or derivatized lipophilic compounds,such as lipid derivatized bleomycin. Antibody targeted liposomes can beconstructed using, for instance, liposomes which incorporate protein A(see Renneisen et al., J. Biol. Chem., 265:16337-16342 (1990) andLeonetti et al., PNAS 87:2448-2451 (1990).

Dosages

For therapeutic applications, the dose administered to a patient, or toa cell which will be introduced into a patient, in the context of thepresent disclosure, should be sufficient to effect a beneficialtherapeutic response in the patient over time. In addition, particulardosage regimens can be useful for determining phenotypic changes in anexperimental setting, e.g., in functional genomics studies, and in cellor animal models. The dose will be determined by the efficacy and K_(d)of the particular ZFP employed, the nuclear volume of the target cell,and the condition of the patient, as well as the body weight or surfacearea of the patient to be treated. The size of the dose also will bedetermined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular compoundor vector in a particular patient.

The maximum therapeutically effective dosage of ZFP for approximately99% binding to target sites is calculated to be in the range of lessthan about 1.5×10⁵ to 1.5×10⁶ copies of the specific ZFP molecule percell. The number of ZFPs per cell for this level of binding iscalculated as follows, using the volume of a HeLa cell nucleus(approximately 1000 μm³ or 10⁻¹² L; Cell Biology, (Altman & Katz, eds.(1976)). As the HeLa nucleus is relatively large, this dosage number isrecalculated as needed using the volume of the target cell nucleus. Thiscalculation also does not take into account competition for ZFP bindingby other sites. This calculation also assumes that essentially all ofthe ZFP is localized to the nucleus. A value of 100×K_(d) is used tocalculate approximately 99% binding of to the target site, and a valueof 10×K_(d) is used to calculate approximately 90% binding of to thetarget site. For this example, K_(d)=25 nM

ZFP + target  site ↔ complexi.e., DNA + protein ↔ DNA:  protein  complex$K_{d} = \frac{\lbrack{DNA}\rbrack \lbrack{protein}\rbrack}{\left\lbrack {{DNA}\text{:}\mspace{14mu} {protein}\mspace{14mu} {complex}} \right\rbrack}$When  50%  of  ZFP  is  bound, K_(d) = [protein]So  when  [protein] = 25  nM  and  the  nucleus  volume  is  10⁻¹²  L$\begin{matrix}{\lbrack{protein}\rbrack = {\left( {25 \times 10^{- 9}\mspace{14mu} {moles}\text{/}L} \right)\left( {10^{- 12}L\text{/}{nucleus}} \right)}} \\{\left( {6 \times 10^{23}\mspace{14mu} {molecules}\text{/}{mole}} \right)} \\{= {15,000\mspace{14mu} {molecules}\text{/}{nucleus}\mspace{14mu} {for}\mspace{14mu} 50\% \mspace{14mu} {binding}}}\end{matrix}$When  99%  target  is  bound; 100 × K_(d) = [protein]100 × K_(d) = [protein] = 2.5  µM(2.5 × 10⁻⁶  moles/L)(10⁻¹²L/nucleus)(6 × 10²³  molecules/mole) = about  1, 500, 000  molecules  per  nucleus  for  99%  binding  of  target  site.

The appropriate dose of an expression vector encoding a ZFP can also becalculated by taking into account the average rate of ZFP expressionfrom the promoter and the average rate of ZFP degradation in the cell.In certain embodiments, a weak promoter such as a wild-type or mutantHSV TK promoter is used, as described above. The dose of ZFP inmicrograms is calculated by taking into account the molecular weight ofthe particular ZFP being employed.

In determining the effective amount of the ZFP to be administered in thetreatment or prophylaxis of disease, the physician evaluates circulatingplasma levels of the ZFP or nucleic acid encoding the ZFP, potential ZFPtoxicities, progression of the disease, and the production of anti-ZFPantibodies. Administration can be accomplished via single or divideddoses.

Pharmaceutical Compositions and Administration

ZFPs and expression vectors encoding ZFPs can be administered directlyto the patient for targeted cleavage and/or recombination, and fortherapeutic or prophylactic applications, for example, cancer, ischemia,diabetic retinopathy, macular degeneration, rheumatoid arthritis,psoriasis, HIV infection, sickle cell anemia, Alzheimer's disease,muscular dystrophy, neurodegenerative diseases, vascular disease, cysticfibrosis, stroke, and the like. Examples of microorganisms that can beinhibited by ZFP gene therapy include pathogenic bacteria, e.g.,chlamydia, rickettsial bacteria, mycobacteria, staphylococci,streptococci, pneumococci, meningococci and conococci, klebsiella,proteus, serratia, pseudomonas, legionella, diphtheria, salmonella,bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, andLyme disease bacteria; infectious fungus, e.g., Aspergillus, Candidaspecies; protozoa such as sporozoa (e.g., Plasmodia), rhizopods (e.g.,Entamoeba) and flagellates (Trypanosoma, Leishmania, Trichomonas,Giardia, etc.); viral diseases, e.g., hepatitis (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HSV-6, HSV-II, CMV, and EBV), HIV, Ebola,adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, poliovirus, rabies virus, andarboviral encephalitis virus, etc.

Administration of therapeutically effective amounts is by any of theroutes normally used for introducing ZFP into ultimate contact with thetissue to be treated. The ZFPs are administered in any suitable manner,preferably with pharmaceutically acceptable carriers. Suitable methodsof administering such modulators are available and well known to thoseof skill in the art, and, although more than one route can be used toadminister a particular composition, a particular route can oftenprovide a more immediate and more effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions that areavailable (see, e.g., Remington's Pharmaceutical Sciences, 17^(th) ed.1985)).

. The ZFPs, alone or in combination with other suitable components, canbe made into aerosol formulations (i.e., they can be “nebulized”) to beadministered via inhalation. Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, and subcutaneousroutes, include aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. The disclosed compositions can beadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically or intrathecally. The formulations ofcompounds can be presented in unit-dose or multi-dose sealed containers,such as ampules and vials. Injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

Applications

The disclosed methods and compositions for targeted cleavage can be usedto induce mutations in a genomic sequence, e.g., by cleaving at twosites and deleting sequences in between, by cleavage at a single sitefollowed by non-homologous end joining, and/or by cleaving at a site soas to remove one or two or a few nucleotides. Targeted cleavage can alsobe used to create gene knock-outs (e.g., for functional genomics ortarget validation) and to facilitate targeted insertion of a sequenceinto a genome (i.e., gene knock-in); e.g., for purposes of cellengineering or protein overexpression. Insertion can be by means ofreplacements of chromosomal sequences through homologous recombinationor by targeted integration, in which a new sequence (i.e., a sequencenot present in the region of interest), flanked by sequences homologousto the region of interest in the chromosome, is inserted at apredetermined target site.

The same methods can also be used to replace a wild-type sequence with amutant sequence, or to convert one allele to a different allele.

Targeted cleavage of infecting or integrated viral genomes can be usedto treat viral infections in a host. Additionally, targeted cleavage ofgenes encoding receptors for viruses can be used to block expression ofsuch receptors, thereby preventing viral infection and/or viral spreadin a host organism. Targeted mutagenesis of genes encoding viralreceptors (e.g., the CCR5 and CXCR4 receptors for HIV) can be used torender the receptors unable to bind to virus, thereby preventing newinfection and blocking the spread of existing infections. Non-limitingexamples of viruses or viral receptors that may be targeted includeherpes simplex virus (HSV), such as HSV-1 and HSV-2, varicella zostervirus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV), HHV6and HHV7. The hepatitis family of viruses includes hepatitis A virus(HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the deltahepatitis virus (HDV), hepatitis E virus (HEV) and hepatitis G virus(HGV). Other viruses or their receptors may be targeted, including, butnot limited to, Picornaviridae (e.g., polioviruses, etc.);Caliciviridae; Togaviridae (e.g., rubella virus, dengue virus, etc.);Flaviviridae; Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae(e.g., rabies virus, etc.); Filoviridae; Paramyxoviridae (e.g., mumpsvirus, measles virus, respiratory syncytial virus, etc.);Orthomyxoviridae (e.g., influenza virus types A, B and C, etc.);Bunyaviridae; Arenaviridae; Retroviradae; lentiviruses (e.g., HTLV-I;HTLV-II; HIV-1 (also known as HTLV-III, LAV, ARV, hTLR, etc.) HIV-II);simian immunodeficiency virus (SW), human papillomavirus (HPV),influenza virus and the tick-borne encephalitis viruses. See, e.g.Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2ndEdition (B. N. Fields and D. M. Knipe, eds. 1991), for a description ofthese and other viruses. Receptors for HIV, for example, include CCR-5and CXCR-4.

In similar fashion, the genome of an infecting bacterium can bemutagenized by targeted DNA cleavage followed by non-homologous endjoining, to block or ameliorate bacterial infections.

The disclosed methods for targeted recombination can be used to replaceany genomic sequence with a homologous, non-identical sequence. Forexample, a mutant genomic sequence can be replaced by its wild-typecounterpart, thereby providing methods for treatment of e.g., geneticdisease, inherited disorders, cancer, and autoimmune disease. In likefashion, one allele of a gene can be replaced by a different alleleusing the methods of targeted recombination disclosed herein.

Exemplary genetic diseases include, but are not limited to,achondroplasia, achromatopsia, acid maltase deficiency, adenosinedeaminase deficiency (OMIM No. 102700), adrenoleukodystrophy, aicardisyndrome, alpha-1 antitrypsin deficiency, alpha-thalassemia, androgeninsensitivity syndrome, apert syndrome, arrhythmogenic rightventricular, dysplasia, ataxia telangictasia, barth syndrome,beta-thalassemia, blue rubber bleb nevus syndrome, canavan disease,chronic granulomatous diseases (CGD), cri du chat syndrome, cysticfibrosis, dercum's disease, ectodermal dysplasia, fanconi anemia,fibrodysplasia ossificans progressive, fragile X syndrome, galactosemis,Gaucher's disease, generalized gangliosidoses (e.g., GM1),hemochromatosis, the hemoglobin C mutation in the 6^(th) codon ofbeta-globin (HbC), hemophilia, Huntington's disease, Hurler Syndrome,hypophosphatasia, Klinefleter syndrome, Krabbes Disease, Langer-GiedionSyndrome, leukocyte adhesion deficiency (LAD, OMIM No. 116920),leukodystrophy, long QT syndrome, Marfan syndrome, Moebius syndrome,mucopolysaccharidosis (MPS), nail patella syndrome, nephrogenic diabetesinsipdius, neurofibromatosis, Neimann-Pick disease, osteogenesisimperfecta, porphyria, Prader-Willi syndrome, progeria, Proteussyndrome, retinoblastoma, Rett syndrome, Rubinstein-Taybi syndrome,Sanfilippo syndrome, severe combined immunodeficiency (SCID), Shwachmansyndrome, sickle cell disease (sickle cell anemia), Smith-Magenissyndrome, Stickler syndrome, Tay-Sachs disease, Thrombocytopenia AbsentRadius (TAR) syndrome, Treacher Collins syndrome, trisomy, tuberoussclerosis, Turner's syndrome, urea cycle disorder, von Hippel-Landaudisease, Waardenburg syndrome, Williams syndrome, Wilson's disease,Wiskott-Aldrich syndrome, X-linked lymphoproliferative syndrome (XLP,OMIM No. 308240).

Additional exemplary diseases that can be treated by targeted DNAcleavage and/or homologous recombination include acquiredimmunodeficiencies, lysosomal storage diseases (e.g., Gaucher's disease,GM1, Fabry disease and Tay-Sachs disease), mucopolysaccahidosis (e.g.Hunter's disease, Hurler's disease), hemoglobinopathies (e.g., sicklecell diseases, HbC, α-thalassemia, β-thalassemia) and hemophilias.

In certain cases, alteration of a genomic sequence in a pluripotent cell(e.g., a hematopoietic stem cell) is desired. Methods for mobilization,enrichment and culture of hematopoietic stem cells are known in the art.See for example, U.S. Pat. Nos. 5,061,620; 5,681,559; 6,335,195;6,645,489 and 6,667,064. Treated stem cells can be returned to a patientfor treatment of various diseases including, but not limited to, SCIDand sickle-cell anemia.

In many of these cases, a region of interest comprises a mutation, andthe donor polynucleotide comprises the corresponding wild-type sequence.Similarly, a wild-type genomic sequence can be replaced by a mutantsequence, if such is desirable. For example, overexpression of anoncogene can be reversed either by mutating the gene or by replacing itscontrol sequences with sequences that support a lower, non-pathologiclevel of expression. As another example, the wild-type allele of theApoAI gene can be replaced by the ApoAI Milano allele, to treatatherosclerosis. Indeed, any pathology dependent upon a particulargenomic sequence, in any fashion, can be corrected or alleviated usingthe methods and compositions disclosed herein.

Targeted cleavage and targeted recombination can also be used to alternon-coding sequences (e.g., regulatory sequences such as promoters,enhancers, initiators, terminators, splice sites) to alter the levels ofexpression of a gene product. Such methods can be used, for example, fortherapeutic purposes, functional genomics and/or target validationstudies.

The compositions and methods described herein also allow for novelapproaches and systems to address immune reactions of a host toallogeneic grafts. In particular, a major problem faced when allogeneicstem cells (or any type of allogeneic cell) are grafted into a hostrecipient is the high risk of rejection by the host's immune system,primarily mediated through recognition of the Major HistocompatibilityComplex (MHC) on the surface of the engrafted cells. The MHC comprisesthe HLA class I protein(s) that function as heterodimers that arecomprised of a common β subunit and variable α subunits. It has beendemonstrated that tissue grafts derived from stem cells that are devoidof HLA escape the host's immune response. See, e.g., Coffman et al. JImmunol 151, 425-35. (1993); Markmann et al. Transplantation 54, 1085-9.(1992); Koller et al. Science 248, 1227-30. (1990). Using thecompositions and methods described herein, genes encoding HLA proteinsinvolved in graft rejection can be cleaved, mutagenized or altered byrecombination, in either their coding or regulatory sequences, so thattheir expression is blocked or they express a non-functional product.For example, by inactivating the gene encoding the common β subunit gene(β2 microglobulin) using ZFP fusion proteins as described herein, HLAclass I can be removed from the cells to rapidly and reliably generateHLA class I null stem cells from any donor, thereby reducing the needfor closely matched donor/recipient MHC haplotypes during stem cellgrafting.

Inactivation of any gene (e.g., the β2 microglobulin gene) can beachieved, for example, by a single cleavage event, by cleavage followedby non-homologous end joining, by cleavage at two sites followed byjoining so as to delete the sequence between the two cleavage sites, bytargeted recombination of a missense or nonsense codon into the codingregion, or by targeted recombination of an irrelevant sequence (i.e., a“stuffer” sequence) into the gene or its regulatory region, so as todisrupt the gene or regulatory region.

Targeted modification of chromatin structure, as disclosed in co-ownedWO 01/83793, can be used to facilitate the binding of fusion proteins tocellular chromatin.

In additional embodiments, one or more fusions between a zinc fingerbinding domain and a recombinase (or functional fragment thereof) can beused, in addition to or instead of the zinc finger-cleavage domainfusions disclosed herein, to facilitate targeted recombination. See, forexample, co-owned U.S. Pat. No. 6,534,261 and Akopian et al. (2003)Proc. Natl. Acad. Sci. USA 100:8688-8691.

In additional embodiments, the disclosed methods and compositions areused to provide fusions of ZFP binding domains with transcriptionalactivation or repression domains that require dimerization (eitherhomodimerization or heterodimerization) for their activity. In thesecases, a fusion polypeptide comprises a zinc finger binding domain and afunctional domain monomer (e.g., a monomer from a dimerictranscriptional activation or repression domain). Binding of two suchfusion polypeptides to properly situated target sites allowsdimerization so as to reconstitute a functional transcription activationor repression domain.

EXAMPLES Example 1 Editing of a Chromosomal hSMC1L1 Gene by TargetedRecombination

The hSMC1L1 gene is the human orthologue of the budding yeast genestructural maintenance of chromosomes 1. A region of this gene encodingan amino-terminal portion of the protein which includes the WalkerATPase domain was mutagenized by targeted cleavage and recombination.Cleavage was targeted to the region of the methionine initiation codon(nucleotides 24-26, FIG. 1), by designing chimeric nucleases, comprisinga zinc finger DNA-binding domain and a FokI cleavage half-domain, whichbind in the vicinity of the codon. Thus, two zinc finger binding domainswere designed, one of which recognizes nucleotides 23-34 (primarycontacts along the top strand as shown in FIG. 1), and the other ofwhich recognizes nucleotides 5-16 (primary contacts along the bottomstrand). Zinc finger proteins were designed as described in co-ownedU.S. Pat. Nos. 6,453,242 and 6,534,261. See Table 2 for the amino acidsequences of the recognition regions of the zinc finger proteins.

Sequences encoding each of these two ZFP binding domains were fused tosequences encoding a FokI cleavage half-domain (amino acids 384-579 ofthe native FokI sequence; Kita et al. (1989) J. Biol. Chem.264:5751-5756), such that the encoded protein contained FokI sequencesat the carboxy terminus and ZFP sequences at the amino terminus. Each ofthese fusion sequences was then cloned in a modified mammalianexpression vector pcDNA3 (FIG. 2).

TABLE 2 Zinc Finger Designs for the hSMC1L1 Gene Target sequence F1 F2F3 F4 CATGGGGTTCCT RSHDLIE TSSSLSR RSDHLST TNSNRIT (SEQ ID NO: 27) (SEQID NO: 28) (SEQ ID NO: 29) (SEQ ID NO: 30) (SEQ ID NO: 31) GCGGCGCCGGCGRSDDLSR RSDDRKT RSEDLIR RSDTLSR (SEQ ID NO: 32) (SEQ ID NO: 33) (SEQ IDNO: 34) (SEQ ID NO: 35) (SEQ ID NO: 36) Note: The zinc finger amino acidsequences shown above (in one-letter code) represent residues −1 through+6, with respect to the start of the alpha-helical portion of each zincfinger. Finger F1 is closest to the amino terminus of the protein, andFinger F4 is closest to the carboxy terminus.

A donor DNA molecule was obtained as follows. First, a 700 base pairfragment of human genomic DNA representing nucleotides 52415936-52416635of the “−” strand of the X chromosome (UCSC human genome release July,2003), which includes the first exon of the human hSMC1L1 gene, wasamplified, using genomic DNA from HEK293 cells as template. Sequences ofprimers used for amplification are shown in Table 3 (“Initial amp 1” and“Initial amp 2”). The PCR product was then altered, using standardoverlap extension PCR methodology (see, e.g., Ho, et al. (1989) Gene77:51-59), resulting in replacement of the sequence ATGGGG (nucleotides24-29 in FIG. 1) to ATAAGAAGC. This change resulted in conversion of theATG codon (methionine) to an ATA codon (isoleucine) and replacement ofGGG (nucleotides 27-29 in FIG. 1) by the sequence AGAAGC, allowingdiscrimination between donor-derived sequences and endogenouschromosomal sequences following recombination. A schematic diagram ofthe hSMC1 gene, including sequences of the chromosomal DNA in the regionof the initiation codon, and sequences in the donor DNA that differ fromthe chromosomal sequence, is given in FIG. 3. The resulting 700 basepair donor fragment was cloned into pCR4BluntTopo, which does notcontain any sequences homologous to the human genome. See FIG. 4.

For targeted mutation of the chromosomal hSMC1L1 gene, the two plasmidsencoding ZFP-FokI fusions and the donor plasmid were introduced into1×10⁶ HEK293 cells by transfection using Lipofectamine 2000®(Invitrogen). Controls included cells transfected only with the twoplasmids encoding the ZFP-FokI fusions, cells transfected only with thedonor plasmid and cells transfected with a control plasmid (pEGFP-N1,Clontech). Cells were cultured in 5% CO₂ at 37° C. At 48 hours aftertransfection, genomic DNA was isolated from the cells, and 200 ng wasused as template for PCR amplification, using one primer complementaryto a region of the gene outside of its region of homology with the donorsequences (nucleotides 52416677-52416701 on the “−” STRAND of the Xchromosome; UCSC July 2003), and a second primer complementary to aregion of the donor molecule into which distinguishing mutations wereintroduced. Using these two primers, an amplification product of 400base pairs will be obtained from genomic DNA if a targeted recombinationevent has occurred. The sequences of these primers are given in Table 3(labeled “chromosome-specific” and “donor-specific,” respectively).Conditions for amplification were: 94° C., 2 min, followed by 40 cyclesof 94° C., 30 sec, 60° C., 1 min, 72° C., 1 min; and a final step of 72°C., 7 min.

The results of this analysis (FIG. 5) indicate that a 400 base pairamplification product (labeled “Chimeric DNA” in the Figure) wasobtained only with DNA extracted from cells which had been transfectedwith the donor plasmid and both ZFP-FokI plasmids.

TABLE 3 Amplification Primers for the hSMC1L1 Gene Initial amp 1AGCAACAACTCCTCCGGGGATC (SEQ ID NO: 37) Initial amp 2TTCCAGACGCGACTCTTTGGC (SEQ ID NO: 38) Chromosome-specificCTCAGCAAGCGTGAGCTCAGGTCTC (SEQ ID NO: 39) Donor-specificCAATCAGTTTCAGGAAGCTTCTT (SEQ ID NO: 40) Outside 1CTCAGCAAGCGTGAGCTCAGGTCTC (SEQ ID NO: 41) Outside 2GGGGTCAAGTAAGGCTGGGAAGC (SEQ ID NO: 42)

To confirm this result, two additional experiments were conducted.First, the amplification product was cloned into pCR4Blunt-Topo(Invitrogen) and its nucleotide sequence was determined. As shown inFIG. 6 (SEQ ID NO: 6), the amplified sequence obtained from chromosomalDNA of cells transfected with the two ZFP-FokI-encoding plasmids and thedonor plasmid contains the AAGAAGC sequence that is unique to the donor(nucleotides 395-401 of the sequence presented in FIG. 6) covalentlylinked to chromosomal sequences not present in the donor molecule(nucleotides 32-97 of FIG. 6), indicating that donor sequences have beenrecombined into the chromosome. In particular, the G→A mutationconverting the initiation codon to an isoleucine codon is observed atposition 395 in the sequence.

In a second experiment, chromosomal DNA from cells transfected only withdonor plasmid, cells transfected with both ZFP-FokI fusion plasmids,cells transfected with the donor plasmid and both ZFP-FokI fusionplasmids or cells transfected with the EGFP control plasmid was used astemplate for amplification, using primers complementary to sequencesoutside of the 700-nucleotide region of homology between donor andchromosomal sequences (identified as “Outside 1” and “Outside 2” inTable 3). The resulting amplification product was purified and used astemplate for a second amplification reaction using the donor-specificand chromosome-specific primers described above (Table 3). Thisamplification yielded a 400 nucleotide product only from cellstransfected with the donor construct and both ZFP-FokI fusionconstructs, a result consistent with the replacement of genomicsequences by targeted recombination in these cells.

Example 2 Editing of a Chromosomal IL2Rγ Gene by Targeted Recombination

The IL-2Rγ gene encodes a protein, known as the “common cytokinereceptor gamma chain,” that functions as a subunit of severalinterleukin receptors (including IL-2R, IL-4R, IL-7R, IL-9R, IL-15R andIL-21R). Mutations in this gene, including those surrounding the 5′ endof the third exon (e.g. the tyrosine 91 codon), can cause X-linkedsevere combined immunodeficiency (SCID). See, for example, Puck et al.(1997) Blood 89:1968-1977. A mutation in the tyrosine 91 codon(nucleotides 23-25 of SEQ ID NO: 7; FIG. 7), was introduced into theIL2Rγ gene by targeted cleavage and recombination. Cleavage was targetedto this region by designing two pairs of zinc finger proteins. The firstpair (first two rows of Table 4) comprises a zinc finger proteindesigned to bind to nucleotides 29-40 (primary contacts along the topstrand as shown in FIG. 7) and a zinc finger protein designed to bind tonucleotides 8-20 (primary contacts along the bottom strand). The secondpair (third and fourth rows of Table 4) comprises two zinc fingerproteins, the first of which recognizes nucleotides 23-34 (primarycontacts along the top strand as shown in FIG. 7) and the second ofwhich recognizes nucleotides 8-16 (primary contacts along the bottomstrand). Zinc finger proteins were designed as described in co-ownedU.S. Pat. Nos. 6,453,242 and 6,534,261. See Table 4 for the amino acidsequences of the recognition regions of the zinc finger proteins.

Sequences encoding the ZFP binding domains were fused to sequencesencoding a FokI cleavage half-domain (amino acids 384-579 of the nativeFokI sequence, Kita et al., supra), such that the encoded proteincontained FokI sequences at the carboxy terminus and ZFP sequences atthe amino terminus. Each of these fusion sequences was then cloned in amodified mammalian expression vector pcDNA3. See FIG. 8 for a schematicdiagram of the constructs.

TABLE 4 Zinc Finger Designs for the IL2Rγ Gene Target sequence F1 F2 F3F4 AACTCGGATA DRSTLIE SSSNLSR RSDDLSK DNSNRIK AT (SEQ ID (SEQ ID NO: 44)(SEQ ID NO: 45) (SEQ ID NO: 46) (SEQ ID NO: 47) NO: 43) TAGAGGaGAAARSDNLSN TSSSRIN RSDHLSQ RNADRKT GG (SEQ ID (SEQ ID NO: 49) (SEQ ID NO:50) (SEQ ID NO: 51) (SEQ ID NO: 52) NO: 48) TACAAGAACT RSDDLSK DNSNRIKRSDALSV DNANRTK CG (SEQ ID NO: 54) (SEQ ID NO: 55) (SEQ ID NO: 56) (SEQID NO: 57) (SEQ ID NO: 53) GGAGAAAGG RSDHLTQ QSGNLAR RSDHLSR (SEQ ID NO:58) (SEQ ID NO: 59) (SEQ ID NO: 60) (SEQ ID NO: 61) Note: The zincfinger amino acid sequences shown above (in one-letter code) representresidues −1 through +6, with respect to the start of the alpha-helicalportion of each zinc finger. Finger F1 is closest to the amino terminusof the protein.

A donor DNA molecule was obtained as follows. First, a 700 base pairfragment of human DNA corresponding to positions 69196910-69197609 onthe “−” strand of the X chromosome (UCSC, July 2003), which includesexon 3 of the of the IL2Rγ gene, was amplified, using genomic DNA fromK562 cells as template. See FIG. 9. Sequences of primers used foramplification are shown in Table 5 (labeled initial amp 1 and initialamp 2). The PCR product was then altered via standard overlap extensionPCR methodology (Ho, et al., supra) to replace the sequenceTACAAGAACTCGGATAAT (SEQ ID NO: 62) with the sequence TAAAAGAATTCCGACAAC(SEQ ID NO: 63). This replacement results in the introduction of a pointmutation at nucleotide 25 (FIG. 7), converting the tyrosine 91 codon TACto a TAA termination codon and enables discrimination betweendonor-derived and endogenous chromosomal sequences followingrecombination, because of differences in the sequences downstream ofcodon 91. The resulting 700 base pair fragment was cloned intopCR4BluntTopo which does not contain any sequences homologous to thehuman genome. See FIG. 10.

For targeted mutation of the chromosomal IL2Rγ gene, the donor plasmid,along with two plasmids each encoding one of a pair of ZFP-FokI fusions,were introduced into 2×10⁶ K652 cells using mixedlipofection/electroporation (Amaxa). Each of the ZFP/FokI pairs (seeTable 4) was tested in separate experiments. Controls included cellstransfected only with two plasmids encoding ZFP-FokI fusions, and cellstransfected only with the donor plasmid. Cells were cultured in 5% CO₂at 37° C. At 48 hours after transfection, genomic DNA was isolated fromthe cells, and 200 ng was used as template for PCR amplification, usingone primer complementary to a region of the gene outside of its regionof homology with the donor sequences (nucleotides 69196839-69196863 onthe “+” strand of the X chromosome; UCSC, July 2003), and a secondprimer complementary to a region of the donor molecule into whichdistinguishing mutations were introduced (see above) and whose sequencetherefore diverges from that of chromosomal DNA. See Table 5 for primersequences, labeled “chromosome-specific” and “donor-specific,”respectively. Using these two primers, an amplification product of 500bp is obtained from genomic DNA in which a targeted recombination eventhas occurred. Conditions for amplification were: 94° C., 2 min, followedby 35 cycles of 94° C., 30 sec, 62° C., 1 min, 72° C., 45 sec; and afinal step of 72° C., 7 min.

The results of this analysis (FIG. 11) indicate that an amplificationproduct of the expected size (500 base pairs) is obtained with DNAextracted from cells which had been transfected with the donor plasmidand either of the pairs of ZFP-FokI-encoding plasmids. DNA from cellstransfected with plasmids encoding a pair of ZFPs only (no donorplasmid) did not result in generation of the 500 bp product, nor did DNAfrom cells transfected only with the donor plasmid.

TABLE 5 Amplification Primers for the IL2Rγ Gene Initial amp 1TGTCGAGTACATGAATTGCACTTGG (SEQ ID NO: 64) Initial amp 2TTAGGTTCTCTGGAGCCCAGGG (SEQ ID NO: 65) Chromosome-specificCTCCAAACAGTGGTTCAAGAATCTG (SEQ ID NO: 66) Donor-specificTCCTCTAGGTAAAGAATTCCGACAAC (SEQ ID NO: 67)

To confirm this result, the amplification product obtained from theexperiment using the second pair of ZFP/FokI fusions was cloned intopCR4Blunt-Topo (Invitrogen) and its nucleotide sequence was determined.As shown in FIG. 12 (SEQ ID NO: 12), the sequence consists of a fusionbetween chromosomal sequences and sequences from the donor plasmid. Inparticular, the G to A mutation converting tyrosine 91 to a stop codonis observed at position 43 in the sequence. Positions 43-58 containnucleotides unique to the donor; nucleotides 32-42 and 59-459 aresequences common to the donor and the chromosome, and nucleotides460-552 are unique to the chromosome. The presence of donor-uniquesequences covalently linked to sequences present in the chromosome butnot in the donor indicates that DNA from the donor plasmid wasintroduced into the chromosome by homologous recombination.

Example 3 Editing of a Chromosomal β-Globin Gene by TargetedRecombination

The human beta globin gene is one of two gene products responsible forthe structure and function of hemoglobin in adult human erythrocytes.Mutations in the beta-globin gene can result in sickle cell anemia. Twozinc finger proteins were designed to bind within this sequence, nearthe location of a nucleotide which, when mutated, causes sickle cellanemia. FIG. 13 shows the nucleotide sequence of a portion of the humanbeta-globin gene, and the target sites for the two zinc finger proteinsare underlined in the sequence presented in FIG. 13. Amino acidsequences of the recognition regions of the two zinc finger proteins areshown in Table 6. Sequences encoding each of these two ZFP bindingdomains were fused to sequences encoding a Fold cleavage half-domain, asdescribed above, to create engineered ZFP-nucleases that targeted theendogenous beta globin gene. Each of these fusion sequences was thencloned in the mammalian expression vector pcDNA3.1 (FIG. 14).

TABLE 6 Zinc Finger Designs for the beta-globin Gene Target sequence F1F2 F3 F4 GGGCAGTAACGG RSDHLSE QSANRTK RSDNLSA RSQNRTR (SEQ ID NO: 68(SEQ ID NO: 69) (SEQ ID NO: 70) (SEQ ID NO: 71) (SEQ ID NO: 72)AAGGTGAACGTG RSDSLSR DSSNRKT RSDSLSA RNDNRKT (SEQ ID NO: 73) (SEQ ID NO:74) (SEQ ID NO: 75) (SEQ ID NO: 76) (SEQ ID NO: 77) Note: The zincfinger amino acid sequences shown above (in one-letter code) representresidues −1 through +6, with respect to the start of the alpha-helicalportion of each zinc finger. Finger F1 is closest to the amino terminusof the protein, and Finger F4 is closest to the carboxy terminus.

A donor DNA molecule was obtained as follows. First, a 700 base pairfragment of human genomic DNA corresponding to nucleotides5212134-5212833 on the “−” strand of Chromosome 11 (BLAT, UCSC HumanGenome site) was amplified by PCR, using genomic DNA from K562 cells astemplate. Sequences of primers used for amplification are shown in Table7 (labeled initial amp 1 and initial amp 2). The resulting amplifiedfragment contains sequences corresponding to the promoter, the first twoexons and the first intron of the human beta globin gene. See FIG. 15for a schematic illustrating the locations of exons 1 and 2, the firstintron, and the primer binding sites in the beta globin sequence. Thecloned product was then further modified by PCR to introduce a set ofsequence changes between nucleotides 305-336 (as shown in FIG. 13),which replaced the sequence

(SEQ ID NO: 78) CCGTTACTGCCCTGTGGGGCAAGGTGAACGTG with (SEQ ID NO: 79)gCGTTAgTGCCCGAATTCCGAtcGTcAACcac (changes in bold).Certain of these changes. (shown in lowercase) were specificallyengineered to prevent the ZFP/FokI fusion proteins from binding to andcleaving the donor sequence, once integrated into the chromosome. Inaddition, all of the sequence changes enable discrimination betweendonor and endogenous chromosomal sequences following recombination. Theresulting 700 base pair fragment was cloned into pCR4-TOPO, which doesnot contain any sequences homologous to the human genome (FIG. 16).

For targeted mutation of the chromosomal beta globin gene, the twoplasmids encoding ZFP-FokI fusions and the donor plasmid(pCR4-TOPO-HBBdonor) were introduced into 1×10⁶ K562 cells bytransfection using Nucleofector™ Solution (Amaxa Biosystems). Controlsincluded cells transfected only with 100 ng (low) or 200 ng (high) ofthe two plasmids encoding the ZFP-FokI fusions, cells transfected onlywith 200 ng (low) or 600 ng (high) of the donor plasmid, cellstransfected with a GFP-encoding plasmid, and mock transfected cells.Cells were cultured in RPMI Medium 1640 (Invitrogen), supplemented with10% fetal bovine serum (FBS) (Hyclone) and 2 mM L-glutamine. Cells weremaintained at 37° C. in an atmosphere of 5% CO₂. At 72 hours aftertransfection, genomic DNA was isolated from the cells, and 200 ng wasused as template for PCR amplification, using one primer complementaryto a region of the gene outside of its region of homology with the donorsequences (nucleotides 5212883-5212905 on the “−” strand of chromosome11), and a second primer complementary to a region of the donor moleculeinto which distinguishing mutations were introduced into the donorsequence (see supra). The sequences of these primers are given in Table7 (labeled “chromosome-specific” and “donor-specific,” respectively).Using these two primers, an amplification product of 415 base pairs willbe obtained from genomic DNA if a targeted recombination event hasoccurred. As a control for DNA loading, PCR reactions were also carriedout using the Initial amp 1 and Initial amp 2 primers to ensure thatsimilar levels of genomic DNA were added to each PCR reaction.Conditions for amplification were: 95° C., 2 min, followed by 40 cyclesof 95° C., 30 sec, 60° C., 45 sec, 68° C., 2 min; and a final step of68° C., 10 min.

The results of this analysis (FIG. 17) indicate that a 415 base pairamplification product was obtained only with DNA extracted from cellswhich had been transfected with the “high” concentration of donorplasmid and both ZFP-FokI plasmids, consistent with targetedrecombination of donor sequences into the chromosomal beta-globin locus.

TABLE 7 Amplification Primers for the human beta globin gene Initial amp1 TACTGATGGTATGGGGCCAAGAG (SEQ ID NO: 80) Initial amp 2CACGTGCAGCTTGTCACAGTGC (SEQ ID NO: 81) Chromosome-specificTGCTTACCAAGCTGTGATTCCA (SEQ ID NO: 82) Donor-specific GGTTGACGATCGGAATTC(SEQ ID NO: 83)

To confirm this result, the amplification product was cloned intopCR4-TOPO (Invitrogen) and its nucleotide sequence was determined. Asshown in FIG. 18 (SEQ ID NO: 14), the sequence consists of a fusionbetween chromosomal sequences not present on the donor plasmid andsequences unique to the donor plasmid. For example, two C→G mutationswhich disrupt ZFP-binding are observed at positions 377 and 383 in thesequence. Nucleotides 377-408 represent sequence obtained from the donorplasmid containing the sequence changes described above; nucleotides73-376 are sequences common to the donor and the chromosome, andnucleotides 1-72 are unique to the chromosome. The covalent linkage ofdonor-specific and chromosome-specific sequences in the genome confirmsthe successful recombination of the donor sequence at the correct locuswithin the genome of K562 cells.

Example 4 ZFP-FokI Linker (ZC Linker) Optimization

In order to test the effect of ZC linker length on cleavage efficiency,a four-finger ZFP binding domain was fused to a FokI cleavagehalf-domain, using ZC linkers of various lengths. The target site forthe ZFP is 5′-AACTCGGATAAT-3′ (SEQ ID NO:84) and the amino acidsequences of the recognition regions (positions −1 through +6 withrespect to the start of the alpha-helix) of each of the zinc fingerswere as follows (wherein F1 is the N-most, and F4 is the C-most zincfinger):

F1: DRSTLIE (SEQ ID NO: 85) F2: SSSNLSR (SEQ ID NO: 86) F3: RSDDLSK (SEQID NO: 87) F4: DNSNRIK (SEQ ID NO: 88)

ZFP-FokI fusions, in which the aforementioned ZFP binding domain and aFokI cleavage half-domain were separated by 2, 3, 4, 5, 6, or 10 aminoacid residues, were constructed. Each of these proteins was tested forcleavage of substrates having an inverted repeat of the ZFP target site,with repeats separated by 4, 5, 6, 7, 8, 9, 12, 15, 16, 17, 22, or 26basepairs.

The amino acid sequences of the fusion constructs, in the region of theZFP-FokI junction (with the ZC linker sequence underlined), are asfollows:

10-residue HTKIHLRQKDAARGSQLV (SEQ ID NO: 89) linker 6-residue linkerHTKIHLRQKGSQLV (SEQ ID NO: 90) 5-residue linker HTKIHLRQGSQLV (SEQ IDNO: 91) 4-residue linker HTKIHLRGSQLV (SEQ ID NO: 92) 3-residue linkerHTKIHLGSQLV (SEQ ID NO: 93) 2-residue linker HTKIHGSQLV (SEQ ID NO: 94)

The sequences of the various cleavage substrates, with the ZFP targetsites underlined, are as follows:

4bp separation CTAGCATTATCCGAGTTACACAACTCGGATAATGCTAGGATCGTAATAGGCTCAATGTGTTGAGCCTATTACGATC (SEQ ID NO: 95) 5bp separationCTAGCATTATCCGAGTTCACACAACTCGGATAATGCTAGGATCGTAATAGGCTCAAGTGTGTTGAGCCTATTACGATC (SEQ ID NO: 96) 6bp separationCTAGGCATTATCCGAGTTCACCACAACTCGGATAATGACTAGGATCCGTAATAGGCTCAAGTGGTGTTGAGCCTATTACTGATC (SEQ ID NO: 97) 7bpseparation CTAGCATTATCCGAGTTCACACACAACTCGGATAATGCTAGGATCGTAATAGGCTCAAGTGTGTGTTGAGCCTATTACGATC (SEQ ID NO: 98) 8bp separationCTAGCATTATCCGAGTTCACCACACAACTCGGATAATGCTAGGATCGTAATAGGCTCAAGTGGTGTGTTGAGCCTATTACGATC (SEQ ID NO: 99) 9bpseparation CTAGCATTATCCGAGTTCACACACACAACTCGGATAATGCTAGGATCGTAATAGGCTCAAGTGTGTGTGTTGAGCCTATTACGATC (SEQ ID NO: 100) 12bpseparation CTAGCATTATCCGAGTTCACCACCAACACAACTCGGATAATGCTAGGATCGTAATAGGCTCAAGTGGTGGTTGTGTTGAGCCTATTACGATC (SEQ ID NO: 101) 15bpseparation CTAGCATTATCCGAGTTCACCACCAACCACACAACTCGGATAATGCTAGGATCGTAATAGGCTCAAGTGGTGGTTGGTGTGTTGAGCCTATTACGATC (SEQ ID NO: 102) 16bpseparation CTAGCATTATCCGAGTTCACCACCAACCACACCAACTCGGATAATGCTAGGATCGTAATAGGCTCAAGTGGTGGTTGGTGTGGTTGAGCCTATTACGATC (SEQ ID NO: 103) 17bpseparation CTAGCATTATCCGAGTTCAACCACCAACCACACCAACTCGGATAATGCTAGGATCGTAATAGGCTCAAGTTGGTGGTTGGTGTGGTTGAGCCTATTACGATC (SEQ ID NO: 104)22bp separation CTAGCATTATCCGAGTTCAACCACCAACCACACCAACACAACTCGGATAATGCTAGGATCGTAATAGGCTCAAGTTGGTGGTTGGTGTGGTTGTGTTGAGCCTATTACGATC (SEQ ID NO:105) 26bp separationCTAGCATTATCCGAGTTCAACCACCAACCACACCAACACCACCAACTCGGATAATGCTAGGATCGTAATAGGCTCAAGTTGGTGGTTGGTGTGGTTGTGGTGGTTGAGCCTATTACGATC (SEQ ID NO:106)

Plasmids encoding the different ZFP-FokI fusion proteins (see above)were constructed by standard molecular biological techniques, and an invitro coupled transcription/translation system was used to express theencoded proteins. For each construct, 200 ng linearized plasmid DNA wasincubated in 20 μL TnT mix and incubated at 30° C. for 1 hour and 45minutes. TnT mix contains 100 μl TnT lysate (Promega, Madison, Wis.)with 4 μl T7 RNA polymerase (Promega)+2 μl Methionine (1 mM)+2.5 μlZnCl₂ (20 mM).

For analysis of DNA cleavage by the different ZFP-FokI fusions, 1 ul ofthe coupled transcription/translation reaction mixture was combined withapproximately 1 ng DNA substrate (end-labeled with ³²P using T4polynucleotide kinase), and the mixture was diluted to a final volume of19 μl with FokI Cleavage Buffer. FokI Cleavage buffer contains 20 mMTris-HCl pH 8.5, 75 mM NaCl, 10 μM ZnCl₂, 1 mM DTT, 5% glycerol, 500μg/ml BSA. The mixture was incubated for 1 hour at 37° C. 6.5 μl of FokIbuffer, also containing 8 mM MgCl₂, was then added and incubation wascontinued for one hour at 37° C. Protein was extracted by adding 10 μlphenol-chloroform solution to each reaction, mixing, and centrifuging toseparate the phases. Ten microliters of the aqueous phase from eachreaction was analyzed by electrophoresis on a 10% polyacrylamide gel.

The gel was subjected to autoradiography, and the cleavage efficiencyfor each ZFP-FokI fusion/substrate pair was calculated by quantifyingthe radioactivity in bands corresponding to uncleaved and cleavedsubstrate, summing to obtain total radioactivity, and determining thepercentage of the total radioactivity present in the bands representingcleavage products.

The results of this experiment are shown in Table 8. This data allowsthe selection of a ZC linker that provides optimum cleavage efficiencyfor a given target site separation. This data also allows the selectionof linker lengths that allow cleavage at a selected pair of targetsites, but discriminate against cleavage at the same or similar ZFPtarget sites that have a separation that is different from that at theintended cleavage site.

TABLE 8 DNA cleavage efficiency for various ZC linker lengths andvarious binding site separations* 6- 10- 2-residue 3-residue 4-residue5-residue residue residue  4 bp 74% 81% 74% 12% 6% 4%  5 bp 61% 89% 92%80% 53% 40%  6 bp 78% 89% 95% 91% 93% 76%  7 bp 15% 55% 80% 80% 70% 80% 8 bp 0% 0% 8% 11% 22% 63%  9 bp 2% 6% 23% 9% 13% 51% 12 bp 8% 12% 22%40% 69% 84% 15 bp 73% 78% 97% 92% 95% 88% 16 bp 59% 89% 100% 97% 90% 86%17 bp 5% 22% 77% 71% 85% 82% 22 bp 1% 3% 5% 8% 18% 58% 26 bp 1% 2% 35%36% 84% 78% *The columns represent different ZFP-FokI fusion constructswith the indicated number of residues separating the ZFP and the FokIcleavage half-domain. The rows represent different DNA substrates withthe indicated number of basepairs separating the inverted repeats of theZFP target site.

For ZFP-FokI fusions with four residue linkers, the amino acid sequenceof the linker was also varied. In separate constructs, the original LRGSlinker sequence (SEQ ID NO:107) was changed to LGGS (SEQ ID NO:108),TGGS (SEQ ID NO:109), GGGS (SEQ ID NO:110), LPGS (SEQ ID NO:111), LRKS(SEQ ID NO:112), and LRWS (SEQ ID NO:113); and the resulting fusionswere tested on substrates having a six-basepair separation betweenbinding sites. Fusions containing the LGGS (SEQ ID NO:108) linkersequence were observed to cleave more efficiently than those containingthe original LRGS sequence(SEQ ID NO:107). Fusions containing theLRKS(SEQ ID NO:112) and LRWS(SEQ ID NO:113) sequences cleaved with lessefficiency than the LRGS sequence(SEQ ID NO:107), while the cleavageefficiencies of the remaining fusions were similar to that of the fusioncomprising the original LRGS sequence(SEQ ID NO:107).

Example 5 Increased Cleavage Specificity Resulting from Alteration ofthe FokI Cleavage Half-Domain in the Dimerization Interface

A pair of ZFP/FokI fusion proteins (denoted 5-8 and 5-10) were designedto bind to target sites in the fifth exon of the IL-2Rγ gene, to promotecleavage in the region between the target sites. The relevant region ofthe gene, including the target sequences of the two fusion proteins, isshown in FIG. 19. The amino acid sequence of the 5-8 protein is shown inFIG. 20, and the amino acid sequence of the 5-10 protein is shown inFIG. 21. Both proteins contain a 10 amino acid ZC linker. With respectto the zinc finger portion of these proteins, the DNA target sequences,as well as amino acid sequences of the recognition regions in the zincfingers, are given in Table 9.

TABLE 9 Zinc Finger Designs for the IL2Rγ Gene Fusion Target sequence F1F2 F3 F4 5-8 ACTCTGTGGAAG RSDNLSE RNAHRIN RSDTLSE ARSTRTT (SEQ ID NO:114) (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 118) NO: 115) NO: 116) NO: 117)5-10 AACACGaAACGTG RSDSLSR DSSNRKT RSDSLSV DRSNRIT (SEQ ID NO: 119) (SEQID (SEQ ID (SEQ ID (SEQ 1D NO: 123) NO: 120) NO: 121) NO: 122) Note: Thezinc finger amino acid sequences shown above (in one-letter code)represent residues −1 through +6, with respect to the start of thealpha-helical portion of each zinc finger. Finger F1 is closest to theamino terminus of the protein.

The ability of this pair of fusion proteins to catalyze specificcleavage of DNA between their target sequences (see FIG. 19) was testedin vitro using a labeled DNA template containing the target sequence andassaying for the presence of diagnostic digestion products. Specificcleavage was obtained when both proteins were used (Table 10, firstrow). However, the 5-10 fusion protein (comprising a wild-type FokIcleavage half-domain) was also capable of aberrant cleavage at anon-target site in the absence of the 5-8 protein (Table 10, secondrow), possibly due to self-dimerization.

Accordingly, 5-10 was modified in its FokI cleavage half-domain byconverting amino acid residue 490 from glutamic acid (E) to lysine (K).(Numbering of amino acid residues in the FokI protein is according toWah et al., supra.) This modification was designed to preventhomodimerization by altering an amino acid residue in the dimerizationinterface. The 5-10 (E490K) mutant, unlike the parental 5-10 protein,was unable to cleave at aberrant sites in the absence of the 5-8 fusionprotein (Table 10, Row 3). However, the 5-10 (E490K) mutant, togetherwith the 5-8 protein, catalyzed specific cleavage of the substrate(Table 10, Row 4). Thus, alteration of a residue in the cleavagehalf-domain of 5-10, that is involved in dimerization, preventedaberrant cleavage by this fusion protein due to self-dimerization. AnE490R mutant also exhibits lower levels of homodimerization than theparent protein.

In addition, the 5-8 protein was modified in its dimerization interfaceby replacing the glutamine (Q) residue at position 486 with glutamicacid (E). This 5-8 (Q486E) mutant was tested for its ability to catalyzetargeted cleavage in the presence of either the wild-type 5-10 proteinor the 5-10 (E490K) mutant. DNA cleavage was not observed when thelabeled substrate was incubated in the presence of both 5-8 (Q486E) andwild-type 5-10 (Table 10, Row 5). However, cleavage was obtained whenthe 5-8 (Q486E) and 5-10 (E490K) mutants were used in combination (Table10, Row 6).

These results indicate that DNA cleavage by a ZFP/FokI fusion proteinpair, at regions other than that defined by the target sequences of thetwo fusion proteins, can be minimized or abolished by altering the aminoacid sequence of the cleavage half-domain in one or both of the fusionproteins.

TABLE 10 DNA cleavage by ZFP/FokI fusion protein pairs containingwild-type and mutant cleavage half-domains ZFP 5-10 binding ZFP 5-8binding domain domain DNA cleavage 1 Wild-type FokI Wild-type FokISpecific 2 Not present Wild-type FokI Non-specific 3 Not present FokIE490K None 4 Wild-type FokI FokI E490K Specific 5 FokI Q486E Wild-typeFokI None 6 FokI Q486E FokI E490K Specific Note: Each row of the tablepresents results of a separate experiment in which ZFP/FokI fusionproteins were tested for cleavage of a labeled DNA substrate. One of thefusion proteins contained the 5-8 DNA binding domain, and the otherfusion protein contained the 5-10 DNA binding domain (See Table 9 andFIG. 19). The cleavage half-domain portion of the fusion proteins was asindicated in the Table. Thus, the entries in the ZFP 5-8 column indicatethe type of FokI cleavage domain fused to ZFP 5-8; and the entries inthe ZFP 5-10 column indicates the type of FokI cleavage domain fused toZFP 5-10. For the FokI cleavage half-domain mutants, the number refersto the amino acid residue in the FokI protein; the letter preceding thenumber refers to the amino acid present in the wild-type protein and theletter following the number denotes the amino acid to which thewild-type residue was changed in generating the modified protein. ‘Notpresent’ indicates that the entire ZFP/FokI fusion protein was omittedfrom that particular experiment. The DNA substrate used in thisexperiment was an approximately 400 bp PCR product containing the targetsites for both ZFP 5-8 and ZFP 5-10. See FIG. 19 for the sequences andrelative orientation of the two target sites.

Example 6 Generation of a Defective Enhanced Green Fluorescent Protein(eGFP) Gene

The enhanced Green Fluorescent Protein (eGFP) is a modified form of theGreen Fluorescent Protein (GFP; see, e.g., Tsien (1998) Ann. Rev.Biochem. 67:509-544) containing changes at amino acid 64 (phe to leu)and 65 (ser to thr). Heim et al. (1995) Nature 373:663-664; Cormack etal. (1996) Gene 173:33-38. An eGFP-based reporter system was constructedby generating a defective form of the eGFP gene, which contained a stopcodon and a 2-bp frameshift mutation. The sequence of the eGFP gene isshown in FIG. 22. The mutations were inserted by overlapping PCRmutagenesis, using the Platinum® Taq DNA Polymerase High Fidelity kit(Invitrogen) and the oligonucleotides GFP-Bam, GFP-Xba, stop sense2, andstop anti2 as primers (oligonucleotide sequences are listed below inTable 11). GFP-Bam and GFP-Xba served as the external primers, while theprimers stop sense2 and stop anti2 served as the internal primersencoding the nucleotide changes. The peGFP-NI vector (BD Biosciences),encoding a full-length eGFP gene, was used as the DNA template in twoseparate amplification reactions, the first utilizing the GFP-Bam andstop anti2 oligonucleotides as primers and the second using the GFP-Xbaand stop sense2 oligonucleotides as primers. This generated twoamplification products whose sequences overlapped. These products werecombined and used as template in a third amplification reaction, usingthe external GFP-Bam and GFP-Xba oligonucleotides as primers, toregenerate a modified eGFP gene in which the sequence GACCACAT (SEQ IDNO: 124) at nucleotides 280-287 was replaced with the sequence TAACAC(SEQ ID NO: 125). The PCR conditions for all amplification reactionswere as follows: the template was initially denatured for 2 minutes at94 degrees and followed by 25 cycles of amplification by incubating thereaction for 30 sec. at 94 degrees C., 45 sec. at 46 degrees C., and 60sec. at 68 degrees C. A final round of extension was carried out at 68degrees C. for 10 minutes. The sequence of the final amplificationproduct is shown in FIG. 23. This 795 bp fragment was cloned into thepCR(R)4-TOPO vector using the TOPO-TA cloning kit (Invitrogen) togenerate the pCR(R)4-TOPO-GFPmut construct.

TABLE 11 Oligonucleotide sequences for GFP Oligo sequence 5′-3′ GFP-BamCGAATTCTGCAGTCGAC (SEQ ID NO: 126) GFP-XbaGATTATGATCTAGAGTCG (SEQ ID NO: 127) stop sense2AGCCGCTACCCCTAACACGAAGCAG (SEQ ID NO: 128) stop anti2CTGCTTCGTGTTAGGGGTAGCGGCT (SEQ ID NO: 129)

Example 7 Design and Assembly of Zinc Finger Nucleases Targeting eGFP

Two three-finger ZFPs were designed to bind a region of the mutated GFPgene (Example 6) corresponding to nucleotides 271-294 (numberingaccording to FIG. 23). The binding sites for these proteins occur inopposite orientation with 6 base pairs separating the two binding sites.See FIG. 23. ZFP 287A binds nucleotides 271-279 on the non-codingstrand, while ZFP 296 binds nucleotides 286-294 on the coding strand.The DNA target and amino acid sequence for the recognition regions ofthe ZFPs are listed below, and in Table 12:

287A: F1 (GCGg) RSDDLTR (SEQ ID NO: 130) F2 (GTA)QSGALAR (SEQ ID NO: 131) F3 (GGG) RSDHLSR (SEQ ID NO: 132) 296S:F1 (GCA) QSGSLTR (SEQ ID NO: 133) F2 (GCA) QSGDLTR (SEQ ID NO: 134)F3 (GAA) QSGNLAR (SEQ ID NO: 135)

TABLE 12 Zinc finger designs for the GFP gene Protein Target sequence F1F2 F3 287A GGGGTAGCGg RSDDLTR QSGALAR RSDHLSR (SEQ ID NO: 136)(SEQ ID NO: 137) (SEQ ID NO: 138) (SEQ ID NO: 139) 296S GAAGCAGCAQSGSLTR QSGDLTR QSGNLAR (SEQ ID NO: 140) (SEQ ID NO: 141)(SEQ ID NO: 142) (SEQ ID NO: 143) Note: The zinc finger amino acidsequences shown above (in one-letter code) represent residues −1 through+6, with respect to the start of the alpha-helical portion of each zincfinger. Finger F1 is closest to the amino terminus of the protein, andFinger F3 is closest to the carboxy terminus.

Sequences encoding these proteins were generated by PCR assembly (e.g.,U.S. Pat. No. 6,534,261), cloned between the KpnI and BamHI sites of thepcDNA3.1 vector (Invitrogen), and fused in frame with the catalyticdomain of the FokI endonuclease (amino acids 384-579 of the sequence ofLooney et al. (1989) Gene 80:193-208). The resulting constructs werenamed pcDNA3.1-GFP287-FokI and pcDNA3.1-GFP296-FokI (FIG. 24).

Example 8 Targeted In Vitro DNA Cleavage by Designed Zinc FingerNucleases

The pCR(R)4-TOPO-GFPmut construct (Example 6) was used to provide atemplate for testing the ability of the 287 and 296 zinc finger proteinsto specifically recognize their target sites and cleave this modifiedform of eGFP in vitro.

A DNA fragment containing the defective eGFP-encoding insert wasobtained by PCR amplification, using the T7 and T3 universal primers andpCR(R)4-TOPO-GFPmut as template. This fragment was end-labeled usingγ-³²P-ATP and T4 polynucleotide kinase. Unincorporated nucleotide wasremoved using a microspin G-50 column (Amersham).

An in vitro coupled transcription/translation system was used to expressthe 287 and 296 zinc finger nucleases described in Example 7. For eachconstruct, 200 ng linearized plasmid DNA was incubated in 20 μA, TnT mixand incubated at 30° C. for 1 hour and 45 minutes. TnT mix contains 100μl TnT lysate (which includes T7 RNA polymerase, Promega, Madison, Wis.)supplemented with 2 μl Methionine (1 mM) and 2.5 μl ZnCl₂ (20 mM).

For analysis of DNA cleavage, aliquots from each of the 287 and 296coupled transcription/translation reaction mixtures were combined, thenserially diluted with cleavage buffer. Cleavage buffer contains 20 mMTris-HCl pH 8.5, 75 mM NaCl, 10 mM MgCl₂, 10 μM ZnCl₂, 1 mM DTT, 5%glycerol, 500 μg/ml BSA. 5 μl of each dilution was combined withapproximately 1 ng DNA substrate (end-labeled with ³²P using T4polynucleotide kinase as described above), and each mixture was furtherdiluted to generate a 20 μl cleavage reaction having the followingcomposition: 20 mM Tris-HCl pH 8.5, 75 mM NaCl, 10 mM MgCl₂, 10 μMZnCl₂, 1 mM DTT, 5% glycerol, 500 μg/ml BSA. Cleavage reactions wereincubated for 1 hour at 37° C. Protein was extracted by adding 10 μlphenol-chloroform solution to each reaction, mixing, and centrifuging toseparate the phases. Ten microliters of the aqueous phase from eachreaction was analyzed by electrophoresis on a 10% polyacrylamide gel.

The gel was subjected to autoradiography, and the results of thisexperiment are shown in FIG. 25. The four left-most lanes show theresults of reactions in which the final dilution of each coupledtranscription/translation reaction mixture (in the cleavage reaction)was 1/156.25, 1/31.25, 1/12.5 and 1/5, respectively, resulting ineffective volumes of 0.032, 0.16, 04. and 1 ul, respectively of eachcoupled transcription/translation reaction. The appearance of two DNAfragments having lower molecular weights than the starting fragment(lane labeled “uncut control” in FIG. 25) is correlated with increasingamounts of the 287 and 296 zinc finger endonucleases in the reactionmixture, showing that DNA cleavage at the expected target site wasobtained.

Example 9 Generation of Stable Cell Lines Containing an IntegratedDefective eGFP Gene

A DNA fragment encoding the mutated eGFP, eGFPmut, was cleaved out ofthe pCR(R)4-TOPO-GFPmut vector (Example 6) and cloned into the HindIIIand NotI sites of pcDNA4/TO, thereby placing this gene under control ofa tetracycline-inducible CMV promoter. The resulting plasmid was namedpcDNA4/TO/GFPmut (FIG. 26). T-Rex 293 cells (Invitrogen) were grown inDulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with10% Tet-free fetal bovine serum (FBS) (HyClone). Cells were plated intoa 6-well dish at 50% confluence, and two wells were each transfectedwith pcDNA4/TO/GFPmut. The cells were allowed to recover for 48 hours,then cells from both wells were combined and split into 10×15-cm² dishesin selective medium, i.e., medium supplemented with 400 ug/ml Zeocin(Invitrogen). The medium was changed every 3 days, and after 10 dayssingle colonies were isolated and expanded further. Each clonal line wastested individually for doxycycline(dox)-inducible expression of theeGFPmut gene by quantitative RT-PCR (TaqMan®).

For quantitative RT-PCR analysis, total RNA was isolated fromdox-treated and untreated cells using the High Pure Isolation Kit (RocheMolecular Biochemicals), and 25 ng of total RNA from each sample wassubjected to real time quantitative RT-PCR to analyze endogenous geneexpression, using TaqMan® assays. Probe and primer sequences are shownin Table 13. Reactions were carried out on an ABI 7700 SDS machine(PerkinElmer Life Sciences) under the following conditions. The reversetranscription reaction was performed at 48° C. for 30 minutes withMultiScribe reverse transcriptase (PerkinElmer Life Sciences), followedby a 10-minute denaturation step at 95° C. Polymerase chain reaction(PCR) was, carried out with AmpliGold DNA polymerase (PerkinElmer LifeSciences) for 40 cycles at 95° C. for 15 seconds and 60° C. for 1minute. Results were analyzed using the SDS version 1.7 software and areshown in FIG. 27, with expression of the eGFPmut gene normalized to theexpression of the human GAPDH gene. A number, of cell lines exhibiteddoxycycline-dependent expression of eGFP; line 18 (T18) was chosen as amodel cell line for further studies.

TABLE 13 Oligonucleotides for mRNA analysis Oligonucleotide SequenceeGFP primer 1 (5T) CTGCTGCCCGACAACCA (SEQ ID NO: 144) eGFP primer 2 (3T)CCATGTGATCGCGCTTCTC (SEQ ID NO: 145) eGFP probe CCCAGTCCGCCCTGAGCAAAGA(SEQ ID NO: 146) GAPDH primer 1 CCATGTTCGTCATGGGTGTGA (SEQ ID NO: 147)GAPDH primer 2 CATGGACTGTGGTCATGAGT (SEQ ID NO: 148) GAPDH probeTCCTGCACCACCAACTGCTTAGCA (SEQ ID NO: 149)

Example 10 Generation of a Donor Sequence for Correction of a DefectiveChromosomal eGFP Gene

A donor construct containing the genetic information for correcting thedefective eGFPmut gene was constructed by PCR. The PCR reaction wascarried out as described above, using the'peGFP-NI vector as thetemplate. To prevent background expression of the donor construct intargeted recombination experiments, the first 12 bp and start codon wereremoved from the donor by PCR using the primers GFPnostart and GFP-Xba(sequences provided in Table 14). The resulting PCR fragment (734 bp)was cloned into the pCR(R)4-TOPO vector, which does not contain amammalian cell promoter, by TOPO-TA cloning to createpCR(R)4-TOPO-GFPdonor5 (FIG. 28). The sequence of the eGFP insert ofthis construct (corresponding to nucleotides 64-797 of the sequenceshown in FIG. 22) is shown in FIG. 29 (SEQ ID NO:20).

TABLE 14 Oligonucleotides for construction of donor moleculeOligonucleotide Sequence 5′-3′ GFPnostartGGCGAGGAGCTGTTCAC (SEQ ID NO: 150) GFP-Xba GATTATGATCTAGAGTCG(SEQ ID NO: 151)

Example 11 Correction of a Mutation in an Integrated Chromosomal eGFPGene by Targeted Cleavage and Recombination

The T18 stable cell line (Example 9) was transfected with one or both ofthe ZFP-FokI expression plasmid (pcDNA3.1-GFP287-FokI andpcDNA3.1-GFP296-FokI, Example 7) and 300 ng of the donor plasmidpCR(R)4-TOPO-GFPdonor5 (Example 10) using LipofectAMINE 2000 Reagent(Invitrogen) in Opti-MEM I reduced serum medium, according to themanufacturer's protocol. Expression of the defective chromosomal eGFPgene was induced 5-6 hours after transfection by the addition of 2 ng/mldoxycycline to the culture medium. The cells were arrested in the G2phase of the cell cycle by the addition, at 24 hours post-transfection,of 100 ng/ml Nocodazole (FIG. 30) or 0.2 uM Vinblastine (FIG. 31). G2arrest was allowed to continue for 24-48 hours, and was then released bythe removal of the medium. The cells were washed with PBS and the mediumwas replaced with DMEM containing tetracycline-free FBS and 2 ng/mldoxycycline. The cells were allowed to recover for 24-48 hours, and genecorrection efficiency was measured by monitoring the number of cellsexhibiting eGFP fluorescence, by fluorescence-activated cell sorting(FACS) analysis. FACS analysis was carried out using a Beckman-CoulterEPICS XL-MCL instrument and System II Data Acquisition and Displaysoftware, version 2.0. eGFP fluorescence was detected by excitation at488 nm with an argon laser and monitoring emissions at 525 nm (x-axis).Background or autofluorescence was measured by monitoring emissions at570 nm (y-axis). Cells exhibiting high fluorescent emission at 525 nmand low emission at 570 nm (region E) were scored positive for genecorrection.

The results are summarized in Table 15 and FIGS. 30 and 31. FIGS. 30 and31 show results in which T18 cells were transfected with thepcDNA3.1-GFP287-FokI and pcDNA3.1-GFP296-FokI plasmids encoding ZFPnucleases and the pCR(R)4-TOPO-GFPdonor5 plasmid, eGFP expression wasinduced with doxycycline, and cells were arrested in G2 with eithernocodazole (FIG. 30) or vinblastine (FIG. 31). Both figures show FACStraces, in which cells exhibiting eGFP fluorescence are represented inthe lower right-hand portion of the trace (identified as Region E, whichis the portion of Quadrant 4 underneath the curve). For transfectedcells that had been treated with nocodazole, 5.35% of the cellsexhibited GFP fluorescence, indicative of correction of the mutantchromosomal eGFP gene (FIG. 30), while 6.7% of cells treated withvinblastine underwent eGFP gene correction (FIG. 31). These results aresummarized, along with additional control experiments, in Rows 1-8 ofTable 15.

In summary, these experiments show that, in the presence of two ZFPnucleases and a donor sequence, approximately 1% of treated cellsunderwent gene correction, and that this level of correction wasincreased 4-5 fold by arresting treated cells in the G2 phase of thecell cycle.

TABLE 15 Correction of a defective chromosomal eGFP gene Percent cellswith Expt. Treatment¹ corrected eGFP gene² 1 300 ng donor only 0.01 2100 ng ZFP 287 + 300 ng donor 0.16 3 100 ng ZFP 296 + 300 ng donor 0.6 450 ng ZFP 287 + 50 ng 1.2 ZFP 296 + 300 ng donor 5 as 4 + 100 ng/mlnocodazole 5.35 6 as 4 + 0.2 uM vinblastine 6.7 7 no donor, no ZFP, 100ng/ml nocodazole 0.01 8 no donor, no ZFP, 0.2 uM vinblastine 0.0 9 100ng ZFP287/Q486E + 300 ng donor 0.0 10 100 ng ZFP296/E490K + 300 ng donor0.01 11 50 ng 287/Q486E + 50 ng 0.62 296/E490K + 300 ng donor 12 as 11 +100 ng/ml nocodazole 2.37 13 as 11 + 0.2 uM vinblastine 2.56 Notes: ¹T18cells, containing a defective chromosomal eGFP gene, were transfectedwith plasmids encoding one or two ZFP nucleases and/or a donor plasmidencoding a nondefective eGFP sequence, and expression of the chromosomaleGFP gene was induced with doxycycline. Cells were optionally arrestedin G2 phase of the cell cycle after eGFP induction. FACS analysis wasconducted 5 days after transfection. ²The number is the percent of totalfluorescence exhibiting high emission at 525 nm and low emission at 570nm (region E of the FACS trace).

Example 12 Correction of a Defective Chromosomal Gene Using Zinc FingerNucleases with Sequence Alterations in the Dimerization Interface

Zinc finger nucleases whose sequences had been altered in thedimerization interface were tested for their ability to catalyzecorrection of a defective chromosomal eGFP gene. The protocol describedin Example 11 was used, except that the nuclease portion of the ZFPnucleases (i.e., the FokI cleavage half-domains) were altered asdescribed in Example 5. Thus, an E490K cleavage half-domain was fused tothe GFP296 ZFP domain (Table 12), and a Q486E cleavage half-domain wasfused to the GFP287 ZFP (Table 12).

The results are shown in Rows 9-11 of Table 15 and indicate that asignificant increase in the frequency of gene correction was obtained inthe presence of two ZFP nucleases having alterations in theirdimerization interfaces, compared to that obtained in the presence ofeither of the nucleases alone. Additional experiments, in which T18cells were transfected with donor plasmid and plasmids encoding the287/Q486E and 296/E490K zinc finger nucleases, then arrested in G2 withnocodazole or vinblastine, showed a further increase in frequency ofgene correction, with over 2% of cells exhibiting eGFP fluorescence,indicative of a corrected chromosomal eGFP gene (Table 15, Rows 12 and13).

Example 13 Effect of Donor Length on Frequency of Gene Correction

In an experiment similar to those described in Example 11, the effect ofthe length of donor sequence on frequency of targeted recombination wastested. T18 cells were transfected with the two ZFP nucleases, and eGFPexpression was induced with doxycycline, as in Example 11. Cells werealso transfected with either the pCR(R)4-TOPO-GFPdonor5 plasmid (FIG.28) containing a 734 bp eGFP insert (FIG. 29) as in Example 11, or asimilar plasmid containing a 1527 bp sequence insert (FIG. 32)homologous to the mutated chromosomal eGFP gene. Additionally, theeffect of G2 arrest with nocodazole on recombination frequency wasassessed.

In a second experiment, donor lengths of 0.7, 1.08 and 1.5 kbp werecompared. T18 cells were transfected with 50 ng of the 287-FokI and296-Fold expression plasmids (Example 7, Table 12) and 500 ng of a 0.7kbp, 1.08 kbp, or 1.5 kbp donors, as described in Example 11. Four daysafter transfection, cells were assayed for correction of the defectiveeGFP gene by FACS, monitoring GFP fluorescence.

The results of these two experiments, shown in Table 16, show thatlonger donor sequence increases the frequency of targeted recombination(and, hence, of gene correction) and confirm that arrest of cells in theG2 phase of the cell cycle also increases the frequency of targetedrecombination.

TABLE 16 Effect of donor length and cell-cycle arrest on targetedrecombination frequency Experiment 1 Nocodazole concentration:Experiment 2 Donor length (kb) 0 ng/ml 100 ng/ml — 0.7 1.41 5.84 1.21.08 not done not done 2.2 1.5 2.16 8.38 2.3 Note: Numbers representpercentage of total fluorescence in Region E of the FACS trace (seeExample 11) which is an indication of the fraction of cells that haveundergone targeted recombination to correct the defective chromosomaleGFP gene.

Example 14 Editing of the Endogenous Human IL-2Rγ Gene by TargetedCleavage and Recombination Using Zinc Finger Nucleases

Two expression vectors, each encoding a ZFP-nuclease targeted to thehuman IL-2Rγ gene, were constructed. Each ZFP-nuclease contained a zincfinger protein-based DNA binding domain (see Table 17) fused to thenuclease domain of the type IIS restriction enzyme FokI (amino acids384-579 of the sequence of Wah et al. (1998) Proc. Natl. Acad. Sci. USA95:10564-10569) via a four amino acid ZC linker (see Example 4). Thenucleases were designed to bind to positions in exon 5 of thechromosomal IL-2Rγ gene surrounding codons 228 and 229 (a mutationalhotspot in the gene) and to introduce a double-strand break in the DNAbetween their binding sites.

TABLE 17 Zinc Finger Designs for exon 5 of the IL2Rγ GeneTarget sequence F1 F2 F3 F4 ACTCTGTGGAAG RSDNLSV RNAHRIN RSDTLSE ARSTRTN(SEQ ID NO: 152) (SEQ ID (SEQ ID (SEQ ID (SEQ ID 5-8G NO: 153) NO: 154)NO: 155) NO: 156) AAAGCGGCTCCG RSDTLSE ARSTRTT RSDSLSK QRSNLKV(SEQ ID NO: 157) 5 (SEQ ID (SEQ ID (SEQ ID (SEQ ID 9D NO: 158) NO: 159)NO: 160) NO: 161) Note: The zinc finger amino acid sequences shown above(in one-letter code) represent residues −1 through +6, with respect tothe start of the alpha-helical portion of each zinc finger. Finger F1 isclosest to the amino terminus of the protein.

The complete DNA-binding portion of each of the chimeric endonucleaseswas as follows:

(SEQ ID NO: 152) Nuclease targeted to ACTCTGTGGAAG (SEQ ID NO: 162)MAERPFQCRICMRNFSRSDNLSVHIRTHTGEKPFACDICGRKFARNAHRINHTKIHTGSQKPFQCRICMRNFSRSDTLSEHIRTHTGEKPFACDICGR KFAARSTRTNHTKIHLRGS(SEQ ID NO: 157) Nuclease targeted to AAAGCGGCTCCG (SEQ ID NO: 163)MAERPFQCRICMRNFSRSDTLSEHIRTHTGEKPFACDICGRKFAARSTRTTHTKIHTGSQKPFQCRICMRNFSRSDSLSKHIRTHTGEKPFACDICG RKFAQRSNLKVHTKIHLRGS

Human embryonic kidney 293 cells were transfected (Lipofectamine 2000;Invitrogen) with two expression constructs, each encoding one of theZFP-nucleases described in the preceding paragraph. The cells were alsotransfected with a donor construct carrying as an insert a 1,543 bpfragment of the IL2Rγ locus corresponding to positions 69195166-69196708of the “minus” strand of the X chromosome (UCSC human genome releaseJuly 2003), in the pCR4Blunt Topo (Invitrogen) vector. The IL-2Rγ insertsequence contained the following two point mutations in the sequence ofexon 5 (underlined):

(SEQ ID NO: 164)  F  R  V  R  S  R  F  N  P  L  C  G  S (SEQ ID NO: 165)TTTCGTGTTCGGAGCCGGTTTAACCCGCTCTGTGGAAGT

The first mutation (CGC→CGG) does not change the amino acid sequence(upper line) and serves to adversely affect the ability of theZFP-nuclease to bind to the donor DNA, and to chromosomal DNA followingrecombination. The second mutation (CCA→CCG) does not change the aminoacid sequence and creates a recognition site for the restriction enzymeBsrBI.

Either 50 or 100 nanograms of each ZFP-nuclease expression construct and0.5 or 1 microgram of the donor construct were used in duplicatetransfections. The following control experiments were also performed:transfection with an expression plasmid encoding the eGFP protein;transfection with donor construct only; and transfection with plasmidsexpressing the ZFP nucleases only. Twenty four hours after transfection,vinblastine (Sigma) was added to 0.2 μM final concentration to onesample in each set of duplicates, while the other remained untreated.Vinblastine affects the cell's ability to assemble the mitotic spindleand therefore acts as a potent G₂ arresting agent. This treatment wasperformed to enhance the frequency of targeting because thehomology-directed double-stranded break repair pathway is more activethan non-homologous end-joining in the G₂ phase of the cell cycle.Following a 48 hr period of treatment with 0.2 μM vinblastine, growthmedium was replaced, and the cells were allowed to recover fromvinblastine treatment for an additional 24 hours. Genomic DNA was thenisolated from all cell samples using the DNEasy Tissue Kit (Qiagen).Five hundred nanograms of genomic DNA from each sample was then assayedfor frequency of gene targeting, by testing for the presence of a newBsrBI site in the chromosomal IL-2Rγ locus, using the assay describedschematically in FIG. 33.

In brief, 20 cycles of PCR were performed using the primers shown inTable 18, each of which hybridizes to the chromosomal IL-2Rγ locusimmediately outside of the region homologous to the 1.5 kb donorsequence. Twenty microcuries each of α-³²P-dCTP and α-³²P-dATP wereincluded in each PCR reaction to allow detection of PCR products. ThePCR reactions were desalted on a G-50 column (Amersham), and digestedfor 1 hour with 10 units of BsrBI (New England Biolabs). The digestionproducts were resolved on a 10% non-denaturing polyacrylamide gel(BioRad), and the gel was dried and autoradiographed (FIG. 34). Inaddition to the major PCR product, corresponding to the 1.55 kbamplififed fragment of the IL2Rγ locus (“wt” in FIG. 34), an additionalband (“rflp” in FIG. 34) was observed in lanes corresponding to samplesfrom cells that were transfected with the donor DNA construct and bothZFP-nuclease constructs. This additional band did not appear in any ofthe control lanes, indicating that ZFP nuclease-facilitatedrecombination of the BsrBI RFLP-containing donor sequence into thechromosome occurred in this experiment.

Additional experiments, in which trace amounts of a RFLP-containingIL-2Rγ DNA sequence was added to human genomic DNA (containing thewild-type IL-2Rγ gene), and the resultant mixture was amplified andsubjected to digestion with a restriction enzyme which cleaves at theRFLP, have indicated that as little as 0.5% RFLP-containing sequence canbe detected quantitatively using this assay.

TABLE 18 Oligonucleotides for analysis of the human IL-2Rγ geneOligonucleotide Sequence Ex5_1.5detF1 GATTCAACCAGACAGATAGAAGG(SEQ ID NO: 166) Ex5_1.5detR1 TTACTGTCTCATCCTTTACTCC (SEQ ID NO: 167)

Example 15 Targeted Recombination at the IL-2Rγ Locus in K562 Cells

K562 is a cell line derived from a human chronic myelogenous leukemia.The proteins used for targeted cleavage were FokI fusions to the 5-8Gand 5-9D zinc finger DNA-binding domains (Example 14, Table 17). Thedonor sequence was the 1.5 kbp fragment of the human IL-2Rγ genecontaining a BsrBI site introduced by mutation, described in Example 14.

K562 cells were cultured in RPMI Medium 1640 (Invitrogen), supplementedwith 10% fetal bovine serum (FBS) (Hyclone) and 2 mM L-glutamine. Allcells were maintained at 37° C. in an atmosphere of 5% CO₂. These cellswere transfected by Nucleofection™ (Solution V, Program T16) (AmaxaBiosystems), according to the manufacturers' protocol, transfecting 2million cells per sample. DNAs for transfection, used in variouscombinations as described below, were a plasmid encoding the 5-8GZFP-FokI fusion endonuclease, a plasmid encoding the 5-9D ZFP-FokIfusion endonuclease, a plasmid containing the donor sequence (describedabove and in Example 14) and the peGFP-N1 vector (BD Biosciences) usedas a control.

In the first experiment, cells were transfected with various plasmids orcombinations of plasmids as shown in Table 19.

TABLE 19 Sample # p-eGFP-N1 p5-8G p5-9D donor vinblastine 1 5 μg — — — —2 — — — 50 μg — 3 — — — 50 μg yes 4 —  10 μg  10 μg — — 5 —   5 μg   5μg 25 μg — 6 —   5 μg   5 μg 25 μg yes 7 — 7.5 μg 7.5 μg 25 μg — 8 — 7.5μg 7.5 μg 25 μg yes 9 — 7.5 μg 7.5 μg 50 μg — 10 — 7.5 μg 7.5 μg 50 μgyes

Vinblastine-treated cells were exposed to 0.2 uM vinblastine at 24 hoursafter transfection for 30 hours. The cells were collected, washed twicewith PBS, and re-plated in growth medium. Cells were harvested 4 daysafter transfection for analysis of genomic DNA.

Genomic DNA was extracted from the cells using the DNEasy kit (Qiagen).One hundred nanograms of genomic DNA from each sample were used in a PCRreaction with the following primers:

(SEQ ID NO: 168) Exon 5 forward: GCTAAGGCCAAGAAAGTAGGGCTAAAG(SEQ ID NO: 169) Exon 5 reverse: TTCCTTCCATCACCAAACCCTCTTG

These primers amplify a 1,669 bp fragment of the X chromosomecorresponding to positions 69195100-69196768 on the “−” strand (UCSChuman genome release July 2003) that contain exon 5 of the IL2Rγ gene.Amplification of genomic DNA which has undergone homologousrecombination with the donor DNA yields a product containing a BsrBIsite; whereas the amplification product of genomic DNA which has notundergone homologous recombination with donor DNA will not contain thisrestriction site.

Ten microcuries each of α-³²PdCTP and α-³²PdATP were included in eachamplification reaction to allow visualization of reaction products.Following 20 cycles of PCR, the reaction was desalted on a Sephadex G-50column (Pharmacia), and digested with 10 Units of BsrBI (New EnglandBiolabs) for 1 hour at 37° C. The reaction was then resolved on a 10%non-denaturing PAGE, dried, and exposed to a PhosphorImager screen.

The results of this experiment are shown in FIG. 35. When cells weretransfected with the control GFP plasmid, donor plasmid alone or the twoZFP-encoding plasmids in the absence of donor, no BsrBI site was presentin the amplification product, as indicated by the absence of the bandmarked “rflp” in the lanes corresponding to these samples in FIG. 35.However, genomic DNA of cells that were transfected with the donorplasmid and both ZFP-encoding plasmids contained the BsrBI siteintroduced by homologous recombination with the donor DNA (band labeled“rflp”). Quantitation of the percentage of signal represented by theRFLP-containing DNA, shown in FIG. 35, indicated that, under optimalconditions, up to 18% of all IL-2Rγ genes in the transfected cellpopulation were altered by homologous recombination.

A second experiment was conducted according to the protocol justdescribed, except that the cells were expanded for 10 days aftertransfection. DNAs used for transfection are shown in Table 20.

TABLE 20 Sample # p-eGFP-N1 p5-8G p5-9D donor vinblastine 1 50 μg — — —— 2 — — — 50 μg — 3 — — — 50 μg yes 4 — 7.5 μg 7.5 μg — — 5 —   5 μg   5μg 25 μg — 6 —   5 μg   5 μg 25 μg yes 7 — 7.5 μg 7.5 μg 50 μg — 8 — 7.5μg 7.5 μg 50 μg yes

Analysis of BsrBI digestion of amplified DNA, shown in FIG. 36, againdemonstrated that up to 18% of IL-2Rγ genes had undergone sequencealteration through homologous recombination, after multiple rounds ofcell division. Thus, the targeted recombination events are stable.

In addition, DNA from transfected cells in this second experiment wasanalyzed by Southern blotting. For this analysis, twelve micrograms ofgenomic DNA from each sample were digested with 100 units EcoRI, 50units BsrBI, and 40 units of DpnI (all from New England Biolabs) for 12hours at 37° C. This digestion generates a 7.7 kbp Eco RI fragment fromthe native IL-2Rγ gene (lacking a BsrBI site) and fragments of 6.7 and1.0 kbp from a chromosomal IL-2Rγ gene whose sequence has been altered,by homologous recombination, to include the BsrBI site. DpnI, amethylation-dependent restriction enzyme, was included to destroy thedam-methylated donor DNA. Unmethylated K562 cell genomic DNA isresistant to DpnI digestion.

Following digestion, genomic DNA was purified by phenol-chloroformextraction and ethanol precipitation, resuspended in TE buffer, andresolved on a 0.8% agarose gel along with a sample of genomic DNAdigested with EcoRI and SphI to generate a size marker. The gel wasprocessed for alkaline transfer following standard procedure and DNA wastransferred to a nylon membrane (Schleicher and Schuell). Hybridizationto the blot was then performed by using a radiolabelled fragment of theIL-2Rγ locus corresponding to positions 69198428-69198769 of the “−”strand of the X chromosome (UCSC human genome July 2003 release). Thisregion of the gene is outside of the region homologous to donor DNA.After hybridization, the membrane was exposed to a PhosphorImager plateand the data quantitated using Molecular Dynamics software. Alterationof the chromosomal IL-2Rγ sequence was measured by analyzing theintensity of the band corresponding to the EcoRI-BsrBI fragment (arrownext to autoradiograph; BsrBI site indicated by filled triangle in themap above the autoradiograph).

The results, shown in FIG. 37, indicate up to 15% of chromosomal IL-2Rγsequences were altered by homologous recombination, thereby confirmingthe results obtained by PCR analysis that the targeted recombinationevent was stable through multiple rounds of cell division. The Southernblot results also indicate that the results shown in FIG. 36 do notresult from an amplification artifact.

Example 16 Targeted Recombination at the IL-2Rγ Locus in CD34-PositiveHematopoietic Stem Cells

Genetic diseases (e.g., severe combined immune deficiency (SCID) andsickle cell anemia) can be treated by homologous recombination-mediatedcorrection of the specific DNA sequence alteration responsible for thedisease. In certain cases, maximal efficiency and stability of treatmentwould result from correction of the genetic defect in a pluripotentcell. To this end, this example demonstrates alteration of the sequenceof the IL-2Rγ gene in human CD34-positive bone marrow cells. CD34⁺ cellsare pluripotential hematopoietic stem cells which give rise to theerythroid, myeloid and lymphoid lineages.

Bone marrow-derived human CD34 cells were purchased from AllCells, LLCand shipped as frozen stocks. These cells were thawed and allowed tostand for 2 hours at 37° C. in an atmosphere of 5%. CO₂ in RPMI Medium1640 (Invitrogen), supplemented with 10% fetal bovine serum (FBS)(Hyclone) and 2 mM L-glutamine. Cell samples (1×10⁶ or 2×10⁶ cells) weretransfected by Nucleofection™ (amaxa biosystems) using the Human CD34Cell Nucleofector™ Kit, according to the manufacturers' protocol. Aftertransfection, cells were cultured in RPMI Medium 1640 (Invitrogen),supplemented with 10% FBS, 2 mM L-glutamine, 100 ng/mlgranulocyte-colony stimulating factor (G-CSF), 100 ng/ml stem cellfactor (SCF), 100 ng/ml thrombopoietin (TPO), 50 ng/ml Flt3 Ligand, and20 ng/ml Interleukin-6 (IL-6). The caspase inhibitor zVAD-FMK(Sigma-Aldrich) was added to a final concentration of 40 uM in thegrowth medium immediately after transfection to block apoptosis.Additional caspase inhibitor was added 48 hours later to a finalconcentration of 20 uM to further prevent apoptosis. These cells weremaintained at 37° C. in an atmosphere of 5% CO₂ and were harvested 3days post-transfection.

Cell numbers and DNAs used for transfection are shown in Table 21.

TABLE 21 Sample # cells p-eGFP-N1¹ Donor² p5-8G³ p5-9D³ 1 1 × 10⁶ 5 ug —— — 2 2 × 10⁶ — 50 ug — — 3 2 × 10⁶ — 50 ug 7.5 ug 7.5 ug ¹This is acontrol plasmid encoding an enhanced green fluorescent protein. ²Thedonor DNA is a 1.5 kbp fragment containing sequences from exon 5 of theIL-2Rγ gene with an introduced BsrBI site (see Example 14). ³These areplasmids encoding FokI fusions with the 5-8 G and 5-9D zinc finger DNAbinding domains (see Table 17).

Genomic DNA was extracted from the cells using the MasterPure DNAPurification Kit (Epicentre). Due to the presence of glycogen in theprecipitate, accurate quantitation of this DNA used as input in the PCRreaction is impossible; estimates using analysis of ethidiumbromide-stained agarose gels indicate that ca. 50 ng genomic DNA wasused in each sample. Thirty cycles of PCR were then performed using thefollowing primers, each of which hybridizes to the chromosomal IL-2Rγlocus immediately outside of the region homologous to the 1.5 kb donor:

(SEQ ID NO: 170) ex5_1.5detF3 GCTAAGGCCAAGAAAGTAGGGCTAAAG(SEQ ID NO: 171) ex5_1.5detR3 TTCCTTCCATCACCAAACCCTCTTG

Twenty microcuries each of α-³²PdCTP and α-³²PdATP were included in eachPCR reaction to allow detection of PCR products. To provide an in-gelquantitation reference, the existence of a spontaneously occurring SNPin exon 5 of the IL-2Rgamma gene in Jurkat cells was exploited: this SNPcreates a RFLP by destroying a MaeII site that is present in normalhuman DNA. A reference standard was therefore created by adding 1 or 10nanograms of normal human genomic DNA (obtained from Clontech, PaloAlto, Calif.) to 100 or 90 ng of Jurkat genomic DNA, respectively, andperforming the PCR as described above. The PCR reactions were desaltedon a G-50 column (Amersham), and digested for 1 hour with restrictionenzyme: experimental samples were digested with 10 units of BsrBI (NewEngland Biolabs); the “reference standard” reactions were digested withMaeII. The digestion products were resolved on a 10% non-denaturing PAGE(BioRad), the gel dried and analyzed by exposure to a PhosphorImagerplate (Molecular Dynamics).

The results are shown in FIG. 38. In addition to the major PCR product,corresponding to the 1.6 kb fragment of the IL2Rγ locus (“wt” in theright-hand panel of FIG. 38), an additional band (labeled “rflp”) wasobserved in lanes corresponding to samples from cells that weretransfected with plasmids encoding both ZFP-nucleases and the donor DNAconstruct. This additional band did not appear in the control lanes,consistent with the idea that ZFP-nuclease assisted gene targeting ofexon 5 of the common gamma chain gene occurred in this experiment.

Although accurate quantitation of the targeting rate is complicated bythe proximity of the RFLP band to the wild-type band; the targetingfrequency was estimated, by comparison to the reference standard (leftpanel), to be between 1-5%.

Example 17 Donor-Target Homology Effects

The effect, on frequency of homologous recombination, of the degree ofhomology between donor DNA and the chromosomal sequence with which itrecombines was examined in T18 cell line, described in Example 9. Thisline contains a chromosomally integrated defective eGFP gene, and thedonor DNA contains sequence changes, with respect to the chromosomalgene, that correct the defect.

Accordingly, the donor sequence described in Example 10 was modified, byPCR mutagenesis, to generate a series of ˜700 bp donor constructs withdifferent degrees of non-homology to the target. All of the modifieddonors contained sequence changes that corrected the defect in thechromosomal eGFP gene and contained additional silent mutations (DNAmutations that do not change the sequence of the encoded protein)inserted into the coding region surrounding the cleavage site. Thesesilent mutations were intended to prevent the binding to, and cleavageof, the donor sequence by the zinc finger-cleavage domain fusions,thereby reducing competition between the intended chromosomal target andthe donor plasmid for binding by the chimeric nucleases. In addition,following homologous recombination, the ability of the chimericnucleases to bind and re-cleave the newly-inserted chromosomal sequences(and possibly stimulating another round of recombination, or causingnon-homologous end joining or other double-strand break-drivenalterations of the genome) would be minimized.

Four different donor sequences were tested. Donor 1 contains 8mismatches with respect to the chromosomal defective eGFP targetsequence, Donor 2 has 10 mismatches, Donor 3 has 6 mismatches, and Donor5 has 4 mismatches. Note that the sequence of donor 5 is identical towild-type eGFP sequence, but contains 4 mismatches with respect to thedefective chromosomal eGFP sequence in the T18 cell line. Table 22provides the sequence of each donor between nucleotides 201-242.Nucleotides that are divergent from the sequence of the defective eGFPgene integrated into the genome of the T18 cell line are shown in boldand underlined. The corresponding sequences of the defective chromosomaleGFP gene (GFP mut) and the normal eGFP gene (GFP wt) are also shown.

TABLE 22 SEQ ID Donor Sequence NO. Donor1 CTTCAGCCGCTA T CC AG A C CACAT 172 GAA A CA A CACGACTTCTT Donor2 CTTCAGCCG G TA T CC AG A C CAC AT173 GAA A CA A CA T GACTTCTT Donor3 CTTCAGCCGCTACCC AG A C CAC AT 174GAA A CAGCACGACTTCTT Donor5 CTTCAGCCGCTACCCC G A C CAC AT 175GAAGCAGCACGACTTCTT GFP mut CTTCAGCCGCTACCCCTAACAC-- 176GAAGCAGCACGACTTCTT GFP wt CTTCAGCCGCTACCCC G A C CAC AT 177GAAGCAGCACGACTTCTT

The T18 cell line was transfected, as described in Example 11, with 50ng of the 287-FokI and 296-FokI expression constructs (Example 7 andTable 12) and 500 ng of each donor construct. FACS analysis wasconducted as described in Example 11.

The results, shown in Table 23, indicate that a decreasing degree ofmismatch between donor and chromosomal target sequence (i.e., increasedhomology) results in an increased frequency of homologous recombinationas assessed by restoration of GFP function.

TABLE 23¹ Percent cells with Donor # mismatches corrected eGFP gene²Donor 2 10 0.45% Donor 1 8 0.53% Donor 3 6 0.89% Donor 5 4 1.56% ¹T18cells, containing a defective chromosomal eGFP gene, were transfectedwith plasmids encoding two ZFP nucleases and with donor plasmidsencoding a nondefective eGFP sequence having different numbers ofsequence mismatches with the chromosomal target sequence. Expression ofthe chromosomal eGFP gene was induced with doxycycline and FACS analysiswas conducted 5 days after transfection. ²The number is the percent oftotal fluorescence exhibiting high emission at 525 nm and low emissionat 570 nm (region E of the FACS trace).

The foregoing results show that levels of homologous recombination areincreased by decreasing the degree of target-donor sequence divergence.Without wishing to be bound by any particular theory or to propose aparticular mechanism, it is noted that greater homology between donorand target could facilitate homologous recombination by increasing theefficiency by which the cellular homologous recombination machineryrecognizes the donor molecule as a suitable template. Alternatively, anincrease in donor homology to the target could also lead to cleavage ofthe donor by the chimeric ZFP nucleases. A cleaved donor could helpfacilitate homologous recombination by increasing the rate of strandinvasion or could aid in the recognition of the cleaved donor end as ahomologous stretch of DNA during homology search by the homologousrecombination machinery. Moreover, these possibilities are not mutuallyexclusive.

Example 18 Preparation of siRNA

To test whether decreasing the cellular levels of proteins involved innon-homologous end joining (NHEJ) facilitates targeted homologousrecombination, an experiment in which levels of the Ku70 protein weredecreased through siRNA inhibition was conducted. siRNA moleculestargeted to the Ku70 gene were generated by transcription of Ku70 cDNAfollowed by cleavage of double-stranded transcript with Dicer enzyme.

Briefly, a cDNA pool generated from 293 and U2OS cells was used in fiveseparate amplification reactions, each using a different set ofamplification primers specific to the Ku70 gene, to generate five poolsof cDNA fragments (pools A-E), ranging in size from 500-750 bp.Fragments in each of these five pools were then re-amplified usingprimers containing the bacteriophage T7 RNA polymerase promoter element,again using a different set of primers for each cDNA pool. cDNAgeneration and PCR reactions were performed using the Superscript ChoicecDNA system and Platinum Taq High Fidelity Polymerase (both fromInvitrogen, Carlsbad, Calif.), according to manufacturers protocols andrecommendations.

Each of the amplified DNA pools was then transcribed in vitro withbacteriophage T7 RNA polymerase to generate five pools (A-E) of doublestranded RNA (dsRNA), using the RNAMAXX in vitro transcription kit(Stratagene, San Diego, Calif.) according to the manufacturer'sinstructions. After precipitation with ethanol, the RNA in each of thepools was resuspended and cleaved in vitro using recombinant Dicerenzyme (Stratagene, San Diego, Calif.) according to the manufacturer'sinstructions. 21-23 bp siRNA products in each of the five pools werepurified by a two-step method, first using a Microspin G-25 column(Amershan), followed by a Microcon YM-100 column (Amicon). Each pool ofsiRNA products was transiently transfected into the T7 cell line usingLipofectamone 2000®.

Western blots to assay the relative effectiveness of the siRNA pools insuppressing Ku70 expression were performed approximately 3 dayspost-transfection. Briefly, cells were lysed and disrupted using RIPAbuffer (Santa Cruz Biotechnology), and homogenized by passing thelysates through a QIAshredder (Qiagen, Valencia, Calif.). The clarifiedlysates were then treated with SDS PAGE sample buffer (with βmercaptoethanol used as the reducing agent) and boiled for 5 minutes.Samples were then resolved on a 4-12% gradient NUPAGE gel andtransferred onto a PVDF membrane. The upper portion of the blot wasexposed to an anti-Ku70 antibody (Santa Cruz sc-5309) and the lowerportion exposed to an anti-TF IIB antibody (Santa Cruz sc-225, used asan input control). The blot was then exposed to horseradishperoxidase-conjugated goat anti-mouse secondary antibody and processedfor electrochemiluminescent (ECL) detection using a kit from PierceChemical Co. according to the manufacturer's instructions.

FIG. 39 shows representative results following transfection of two ofthe siRNA pools (pools D and E) into T7 cells. Transfection with 70 ngof siRNA E results in a significant decrease in Ku70 protein levels(FIG. 39, lane 3).

Example 19 Increasing the Frequency of Homologous Recombination byInhibition of Expression of a Protein Involved in Non-Homologous EndJoining

Repair of a double-stranded break in genomic DNA can proceed along twodifferent cellular pathways; homologous recombination (HR) ornon-homologous end joining (NHEJ). Ku70 is a protein involved in NHEJ,which binds to the free DNA ends resulting from a double-stranded breakin genomic DNA. To test whether lowering the intracellular concentrationof a protein involved in NHEJ increases the frequency of HR, smallinterfering RNAs (siRNAs), prepared as described in Example 18, wereused to inhibit expression of Ku70 mRNA, thereby lowering levels of Ku70protein, in cells co-transfected with donor DNA and with plasmidsencoding chimeric nucleases.

For these experiments, the T7 cell line (see Example 9 and FIG. 27) wasused. These cells contain a chromosomally-integrated defective eGFPgene, but have been observed to exhibit lower levels of targetedhomologous recombination than the T18 cell line used in Examples 11-13.

T7 cells were transfected, as described in Example 11, with either 70 or140 ng of one of two pools of dicer product targeting Ku70 (see Example18). Protein blot analysis was performed on extracts derived from thetransfected cells to determine whether the treatment of cells with siRNAresulted in a decrease in the levels of the Ku70 protein (see previousExample). FIG. 39 shows that levels of the Ku70 protein were reduced incells that had been treated with 70 ng of siRNA from pool E.

Separate cell samples in the same experiment were co-transfected with 70or 140 ng of siRNA (pool D or pool E) along with 50 ng each of the287-Fold and 296-Fold expression constructs (Example 7 and Table 12) and500 ng of the 1.5 kbp GFP donor (Example 13), to determine whetherlowering Ku70 levels increased the frequency of homologousrecombination. The experimental protocol is described in Table 24.Restoration of eGFP activity, due to homologous recombination, wasassayed by FACS analysis as described in Example 11.

TABLE 24 Expt. # Donor¹ ZFNs² SiRNA³ % correction⁴ 1 500 ng — — 0.05 2 —50 ng each — 0.01 3 500 ng 50 ng each — 0.79 4 500 ng 50 ng each  70 ngpool D 0.68 5 500 ng 50 ng each 140 ng pool D 0.59 6 500 ng 50 ng each 70 ng pool E 1.25 7 500 ng 50 ng each 140 ng pool E 0.92 ¹A plasmidcontaining a 1.5 kbp sequence encoding a functional eGFP protein whichis homologous to the chromosomally integrated defective eGFP gene²Plasmids encoding the eGFP-targeted 287 and 296 zinc fingerprotein/FokI fusion endonucleases ³See Example 18 ⁴Percent of totalfluorescence exhibiting high emission at 525 nm and low emission at 570nm (region E of the FACS trace, see Example 11).

The percent correction of the defective eGFP gene in the transfected T7cells (indicative of the frequency of targeted homologous recombination)is shown in the right-most column of Table 24. The highest frequency oftargeted recombination is observed in Experiment 6, in which cells weretransfected with donor DNA, plasmids encoding the two eGFP-targetedfusion nucleases and 70 ng of siRNA Pool E. Reference to Example 18 andFIG. 39 indicates that 70 ng of Pool E siRNA significantly depressedKu70 protein levels. Thus, methods that reduce cellular levels ofproteins involved in NHEJ can be used as a means of facilitatinghomologous recombination.

Example 20 Zinc Finger-FokI Fusion Nucleases Targeted to the Humanβ-Globin Gene

A number of four-finger zinc finger DNA binding domains, targeted to thehuman β-globin gene, were designed and plasmids encoding each zincfinger domain, fused to a FokI cleavage half-domain, were constructed.Each zinc finger domain contained four zinc fingers and recognized a 12bp target site in the region of the human β-globin gene encoding themutation responsible for Sickle Cell Anemia. The binding affinity ofeach of these proteins to its target sequence was assessed, and fourproteins exhibiting strong binding (sca-r29b, sca-36a, sca-36b, andsca-36c) were used for construction of FokI fusion endonucleases.

The target sites of the ZFP DNA binding domains, aligned with thesequence of the human β-globin gene, are shown below. The translationalstart codon (ATG) is in bold and underlined, as is the A-T substitutioncausing Sickle Cell Anemia.

sca-36a (SEQ ID NO: 178) GAAGTCTGCCGT sca-36b (SEQ ID NO: 179)GAAGTCtGCCGTT sca-36c (SEQ ID NO: 180) GAAGTCtGCCGTTCAAACAGACACC ATGGTGCATCTGACTCC TG T GGAGAAGTCTGCCGTTACTG (SEQ ID NO: 181)GTTTGTCTGTGGTACCACGTAGACTGAGGAC A CCTCTTCAGACG GCAATGAC sca-r29b(SEQ ID NO: 182) ACGTAGaCTGAGG

Amino acid sequences of the recognition regions of the zinc fingers inthese four proteins are shown in Table 25. The complete amino acidsequences of these zinc finger domains are shown in FIG. 40. The sca-36adomain recognizes a target site having 12 contiguous nucleotides (shownin upper case above), while the other three domain recognize a thirteennucleotide sequence consisting of two six-nucleotide target sites (shownin upper case) separated by a single nucleotide (shown in lower case).Accordingly, the sca-r29b, sca-36b and sca-36c domains contain anon-canonical inter-finger linker having the amino acid sequenceTGGGGSQKP (SEQ ID NO:183) between the second and the third of their fourfingers.

TABLE 25 ZFP F1 F2 F3 F4 sca-r29b QSGDLTR TSANLSR DRSALSR QSGHLSR(SEQ ID NO: 184) (SEQ ID NO: 185) (SEQ ID NO: 186) (SEQ ID NO: 187)sca-36a RSQTRKT QKRNRTK DRSALSR QSGNLAR (SEQ ID NO: 188)(SEQ ID NO: 189) (SEQ ID NO: 190) (SEQ ID NO: 191) sca-36b TSGSLSRDRSDLSR DRSALSR QSGNLAR (SEQ ID NO: 192) (SEQ ID NO: 193)(SEQ ID NO: 194) (SEQ ID NO: 195) sca-36c TSSSLSR DRSDLSR DRSALSRQSGNLAR (SEQ ID NO: 196) (SEQ ID NO: 197) (SEQ ID NO: 198)(SEQ ID NO: 199)

Example 21 In Vitro Cleavage of a DNA Target Sequence byβ-Globin-Targeted ZFP/FokI Fusion Endonucleases

Fusion proteins containing a FokI cleavage half-domain and one the fourZFP DNA binding domains described in the previous example were testedfor their ability to cleave DNA in vitro with the predicted sequencespecificity. These ZFP domains were cloned into the pcDNA3.1 expressionvector via KpnI and BamHI sites and fused in-frame to the FokI cleavagedomain via a 4 amino acid ZC linker, as described above. A DNA fragmentcontaining 700 bp of the human β-globin gene was cloned from genomic DNAobtained from K562 cells. The isolation and sequence of this fragmentwas described in Example 3, supra.

To produce fusion endonucleases (ZFNs) for the in vitro assay, circularplasmids encoding FokI fusions to sca-r29b, sca-36a, sca-36b, andsca-36c protein were incubated in an in vitro transcription/translationsystem. See Example 4. A total of 2 ul of the TNT reaction (2 ul of asingle reaction when a single protein was being assayed or 1 ul of eachreaction when a pair of proteins was being assayed) was added to 13 ulof the cleavage buffer mix and 3 ul of labeled probe (˜1 ng/ul). Theprobe was end-labeled with ³²P using polynucleotide kinase. Thisreaction was incubated for 1 hour at room temperature to allow bindingof the ZFNs. Cleavage was stimulated by the addition of 8 ul of 8 mMMgCl₂, diluted in cleavage buffer, to a final concentration ofapproximately 2.5 mM. The cleavage reaction was incubated for 1 hour at37° C. and stopped by the addition of 11 ul of phenol/chloroform. TheDNA was isolated by phenol/chloroform extraction and analyzed by gelelectrophoresis, as described in Example 4. As a control, 3 ul of probewas analyzed on the gel to mark the migration of uncut DNA (labeled “U”in FIG. 41).

The results are shown in FIG. 41. Incubation of the target DNA with anysingle zinc finger/FokI fusion resulted in no change in size of thetemplate DNA. However, the combination of the sca-r29b nuclease witheither of the sca-36b or sca-36c nucleases resulted in cleavage of thetarget DNA, as evidenced by the presence of two shorter DNA fragments(rightmost two lanes of FIG. 41).

Example 22 ZFP/FokI Fusion Endonucleases, Targeted to the β-Globin Gene,Tested in a Chromosomal GFP Reporter System

A DNA fragment containing the human β-globin gene sequence targeted bythe ZFNs described in Example 20 was synthesized and cloned into a SpeIsite in an eGFP reporter gene thereby, disrupting eGFP expression. Thefragment contained the following sequence, in which the nucleotideresponsible for the sickle cell mutation is in bold and underlined):

(SEQ ID NO: 200) CTAGACACCATGGTGCATCTGACTCCTG T GGAGAAGTCTGCCGTTACTGCCCTAG

This disrupted eGFP gene containing inserted β-globin sequences wascloned into pcDNA4/TO (Invitrogen, Carlsbad, Calif.) using the HindIIIand NotI sites, and the resulting vector was transfected into HEK293TRex cells (Invitrogen). Individual stable clones were isolated andgrown up, and the clones were tested for targeted homologousrecombination by transfecting each of the sca-36 proteins (sca-36a,sca-36b, sca-36c) paired with sca-29b (See Example 20 and Table 25 forsequences and binding sites of these chimeric nucleases). Cells weretransfected with 50 ng of plasmid encoding each of the ZFNs and with 500ng of the 1.5-kb GFP Donor (Example 13). Five days after transfection,cells were tested for homologous recombination at the inserted defectiveeGFP locus. Initially, cells were examined by fluorescence microscopyfor eGFP function. Cells exhibiting fluorescence were then analyzedquantitatively using a FACS assay for eGFP fluorescence, as described inExample 11.

The results showed that all cell lines transfected with sca-29b andsca-36a were negative for eGFP function, when assayed by fluorescencemicroscopy. Some of the lines transfected with sca-29b paired witheither sca-36b or sca-36c were positive for eGFP expression, whenassayed by fluorescence microscopy, and were therefore further analyzedby FACS analysis. The results of FACS analysis of two of these lines areshown in Table 26, and indicate that zinc finger nucleases targeted toβ-globin sequences are capable of catalyzing sequence-specificdouble-stranded DNA cleavage to facilitate homologous recombination inliving cells.

TABLE 26 DNA transfected: Cell line sca-29b sca-36a sca-36b sca-36c %corr.¹ #20 + + 0 + + 0.08 + + 0.07 #40 + + 0 + + 0.18 + + 0.12 ¹Percentof total fluorescence exhibiting high emission at 525 nm and lowemission at 570 nm (region E of the FACS trace, see Example 11).

Example 23 Effect of Transcription Level on Targeted HomologousRecombination

Since transcription of a chromosomal DNA sequence involves alterationsin its chromatin structure (generally to make the transcribed sequencesmore accessible), it is possible that an actively transcribed gene mightbe a more favorable substrate for targeted homologous recombination.This idea was tested using the T18 cell line (Example 9) which containschromosomal sequences encoding a defective eGFP gene whose transcriptionis under the control of a doxycycline-inducible promoter.

Separate samples of T18 cells were transfected with plasmids encodingthe eGFP-targeted 287 and 296 zinc finger/FokI fusion proteins (Example7) and a 1.5 kbp donor DNA molecule containing sequences that correctthe defect in the chromosomal eGFP gene (Example 9). Five hours aftertransfection, transfected cells were treated with differentconcentrations of doxycycline, then eGFP mRNA levels were measured 48hours after addition of doxycycline. eGFP fluorescence at 520 nm(indicative of targeted recombination of the donor sequence into thechromosome to replace the inserted β-globin sequences) was measured byFACS at 4 days after transfection.

The results are shown in FIG. 42. Increasing steady-state levels of eGFPmRNA normalized to GAPDH mRNA (equivalent, to a first approximation, tothe rate of transcription of the defective chromosomal eGFP gene) areindicated by the bars. The number above each bar indicate the percent ofcells exhibiting eGFP fluorescence. The results show that increasingtranscription rate of the target gene is accompanied by higherfrequencies of targeted recombination. This suggests that targetedactivation of transcription (as disclosed, e.g. in co-owned U.S. Pat.Nos. 6,534,261 and 6,607,882) can be used, in conjunction with targetedDNA cleavage, to stimulate targeted homologous recombination in cells.

Example 24 Generation of a Cell Line Containing a Mutation in the IL-2RγGene

K562 cells were transfected with plasmids encoding the 5-8GL0 and the5-9DL0 zinc finger nucleases (ZFNs) (see Example 14; Table 17) and witha 1.5 kbp DraI donor construct. The DraI donor is comprised of asequence with homology to the region encoding the 5^(th) exon of theIL2Rγ gene, but inserts an extra base between the ZFN-binding sites tocreate a frameshift and generate a DraI site.

24 hours post-transfection, cells were treated with 0.2 uM vinblastine(final concentration) for 30 hours. Cells were washed three times withPBS and re-plated in medium. Cells were allowed to recover for 3 daysand an aliquot of cells were removed to perform a PCR-based RFLP assay,similar to that described in Example 14, testing for the presence of aDraI site. It was determined the gene correction frequency within thepopulation was approximately 4%.

Cells were allowed to recover for an additional 2 days and 1600individual cells were plated into 40×96-well plates in 100 ul of medium.

The cells are grown for about 3 weeks, and cells homozygous for the DraImutant phenotype are isolated. The cells are tested for genomemodification (by testing for the presence of a DraI site in exon 5 ofthe IL-2Rγ gene) and for levels of IL-2Rγ mRNA (by real-time PCR) andprotein (by Western blotting) to determine the effect of the mutation ongene expression. Cells are tested for function by FACS analysis.

Cells containing the DraI frameshift mutation in the IL-2Rγ gene aretransfected with plasmids encoding the 5-8GL0 and 5-9DL0 fusion proteinsand a 1.5 kb BsrBI donor construct (Example 14) to replace the DraIframeshift mutation with a sequence encoding a functional protein.Levels of homologous recombination greater than 1% are obtained in thesecells, as measured by assaying for the presence of a BsrBI site asdescribed in Example 14. Recovery of gene function is demonstrated bymeasuring mRNA and protein levels and by FACS analysis.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference, in their entireties, for all purposes.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

1. A method of altering a non-coding sequence in a cell containing aregion of interest in cellular chromatin, the method comprising: (a)selecting first and second nucleotide sequences in the region ofinterest; (b) engineering first and second zinc finger binding domainsto bind to the first and second nucleotide sequences; and (c) expressinga first fusion protein in the cell, the first fusion protein comprisinga first engineered zinc finger binding domain and a first cleavagehalf-domain; (d) expressing a second fusion protein in the cell, thesecond fusion protein comprising a second zinc finger binding domain anda second cleavage half-domain wherein the first and second fusionproteins cleave cellular chromatin and alter the non-coding sequence inthe region of interest.
 2. The method of claim 1, wherein the cleavagehalf-domains of the first and second fusion proteins are from the sameendonuclease.
 3. The method of claim 2 wherein the endonuclease is aType IIS restriction endonuclease.
 4. The method of claim 3 wherein theType IIS restriction endonuclease is Fok I.
 5. The method of claim 1,wherein cleavage occurs between the first and second nucleotidesequences.
 6. The method of claim 1, wherein non-coding sequences in theregion of interest are deleted.
 7. The method of claim 1, wherein thealteration is selected from the group consisting of one or morenucleotide substitutions, one or more nucleotide deletions and one ormore nucleotide insertions.
 8. The method of claim 1, wherein thenon-coding sequence is a transcriptional regulatory sequence.
 9. Themethod of claim 1 wherein, the first and second fusion proteins areexpressed from different polynucleotides.
 10. The method of claim 1wherein, the first and second fusion proteins are expressed from thesame polynucleotide.
 11. The method of claim 1, wherein the cell is aeukaryotic cell.
 12. The method of claim 11, wherein the cell is a plantcell.
 13. The method of claim 11, wherein the cell is a mammalian cell.14. A method of introducing an exogenous sequence into a non-codingregion of interest of cellular chromatin of a cell, the methodcomprising, altering the non-coding sequence in the cell according tothe method of claim 1; and introducing a polynucleotide comprising theexogenous sequence into the cell, such that when the cellular chromatinis cleaved in the cell, the exogenous sequence is integrated into thenon-coding region of interest.
 15. The method of claim 14, wherein thecell is a plant cell.
 16. The method of claim 14, wherein the cell is amammalian cell.
 17. A eukaryotic cell made by the method of claim
 1. 18.The cell of claim 17, wherein the cell is a plant cell.
 19. The cell ofclaim 17, wherein the cell is a mammalian cell.
 20. A geneticallymodified organism made by the method of claim
 14. 21. The geneticallymodified organism of claim 20, wherein the organism is a plant.
 22. Thegenetically modified organism of claim 20, wherein the organism is amammal.